Isolation and Characterization of Bacteria in a Toluene-Producing Enrichment Culture Derived from Contaminated Groundwater at a Louisiana Superfund Site

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August 2020

Madison Mikes Louisiana State University and Agricultural and Mechanical College

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Recommended Citation Mikes, Madison, "Isolation and Characterization of Bacteria in a Toluene-Producing Enrichment Culture Derived from Contaminated Groundwater at a Louisiana Superfund Site" (2020). LSU Master's Theses. 5206. https://digitalcommons.lsu.edu/gradschool_theses/5206

This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. ISOLATION AND CHARACTERIZATION OF BACTERIA IN A TOLUENE- PRODUCING ENRICHMENT CULTURE DERIVED FROM CONTAMINATED GROUNDWATER AT A LOUISIANA SUPERFUND SITE

A Thesis

Submitted to the Graduate Faculty of the Louisiana State University and Agriculture and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science

The Department of Civil and Environmental Engineering

by Madison Colleen Mikes B.S., Louisiana State University, 2018 December 2020

ACKNOWLEDGEMENTS

I would like to take the time to thank all of those who have supported and assisted me during my graduate program. First and foremost, I would like to thank Dr. Bill Moe for all of the time he has spent teaching me and mentoring me through my thesis work. I have learned so much in the past two years, and I am so grateful that I had the opportunity to participate in this research. I would also like to thank the members of my thesis committee Dr. John Pardue and Dr.

Gary King for their contributions to my thesis.

I would like to acknowledge my laboratory peers that have supported me during the past two years. My thanks goes to Morgan, Alexis, Sam, Emily, Chi, Maura, and Shane for assisting me with my project as well as being there to discuss ideas and plans for new experiments. All of the continuous support kept me motivated and excited to go to the lab every day. Additionally, thank you to Sam Reynolds for developing the toluene-producing enrichment cultures that were used for my cultivation experiments.

I am so grateful for the support of my family and friends. Thank you Mom, Dad, and Kel for motivating me to do my best work. Matt and Alison, thank you for being there for me and letting me vent to you when I was stressed and overwhelmed. I appreciate all of the advice and support. You made my experience in Baton Rouge so much more memorable.

I also would like to thank Dr. Ying Xiao for assistance with scanning electron microscopy. Finally, I would like to thank the Louisiana Board of Regents and a consortium of petrochemical companies for financial support of my research.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... ii

LIST OF TABLES ...... v

LIST OF FIGURES ...... vii

ABSTRACT ...... viii

CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ...... 1 1.1. Introduction ...... 1 1.2. Literature Review ...... 1 1.3. Research Objectives ...... 8 1.4. Thesis Organization ...... 9

CHAPTER 2. CULTIVATION AND ISOLATION OF BACTERIA DERIVED FROM A TOLUENE-PRODUCING ENRICHMENT CULTURE ON SOLID MEDIA ...... 10 2.1. Introduction ...... 10 2.2. Toluene-Producing Enrichment Cultures ...... 10 2.2. Medium Preparation and Inoculation ...... 14 2.3. Stock Solution Preparation ...... 18 2.4. Experimental Design for Isolation and Identification of Bacteria ...... 20 2.5. Results and Discussion...... 30 2.6. Conclusion ...... 41

CHAPTER 3. CHARACTERIZATION OF NOVEL AZOSPIRA ISOLATES ...... 42 3.1. Background of the genus Azospira ...... 42 3.2. Materials and Methods ...... 44 3.3. Media Preparation ...... 45 3.4. Comparative Testing ...... 46 3.5. Results ...... 56 3.6. Discussion ...... 66

CHAPTER 4. CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH.. 70 4.1. Conclusions ...... 70 4.2. Recommendations for future research ...... 72

APPENDIX A. REPRESENTATIVE SEQUENCES FROM ISOLATES ...... 78

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APPENDIX B. STRAIN AZ-3 ASSEMBELED SEQUENCE ...... 83

REFERENCES ...... 84

VITA ...... 91

LIST OF TABLES

Table 1.1. Phylogenetically related subdivision 1 Acidobacteria based on 16S rRNA gene sequences and maximum likelihood algorithm presented in Myers and King (2016)……………6 Table 2.1. Groundwater well location from which the SR enrichment culture originated [data from Moe et al. (2018)]……………………………………………...…………………….10 Table 2.2. Volatile organic compound (VOC) concentrations measured in groundwater collected for establishment of enrichment cultures from well SP022 on April 24, 2017 [222 days after the fourth molasses injection, data from Moe et al. (2018)] ……………………12 Table 2.3. Geochemical parameters measured in groundwater collected for establishment of enrichment cultures from well SP022 on April 24, 2017 [222 days after fourth molasses injection, data from Moe et al. (2018)]…………………………………………………………..12

Table 2.4. Growth Media used to Isolate Colonies from the Toluene-Producing Enrichment Cultures…………………………………………………………………………………………..17

Table 2.5. Universal bacterial Primers Used for Isolates…………………………….…………..23

Table 2.6. Universal and “Koribacter”-specific Primer Sets Used for Plate Wash PCR….….….25

Table 2.7. Expected Amplicon Size from Primer Sets…………………………………………..26

Table 2.8. Additional Primers used for Acidobacteria-targeted PCR…………………………...26

Table 2.9. Thermal Program for all Primer Sets………………………………………………....28

Table 2.10. Strains Inoculated into Liquid PA2D to Assess for Toluene Production…………...29

Table 2.11. Dilution Level of Colonies Isolated after Various Days of Incubation……………..31

Table 2.12. Closest Representatives to Grouped Isolates Based on the Blastn Algorithm Using the NCBI Database…………..……………………………………………………………34

Table 2.13. Closest Validly Published Bacterial Species to Grouped Isolates as Determined from EZ-Taxon……………………...…………………………………………………………...35

Table 2.14. Top Blastn Match for Positive Control DNA Amplified with Acidobacteria-specific Primer Sets…...………………………………………………………….39 Table 3.1. Pairwise similarity between strains Az-1, Az-2, and Az-3 and the most closely related type strains in the EZBioCloud database………………………………………………...56 Table 3.2. Substrate Utilization Profile for all Strains. (+) positive, (-) negative………..………61

Table 3.3. Presence of Cellular Fatty Acids in all Strains (those that represent <0.5% in all strains were omitted). All data was reported in this study except those marked with an “*” which was reported by Bae et al. (2007)…………………………………………………….65 Table 3.4. Phenotypic Differences between the new isolates and previously described Azospira spp…………………………………………………………………………….………..69

LIST OF FIGURES

Figure 2.1. Percent Abundance of Organisms from all Cultured Bacteria by Group (see Table 2.12. and 2.13. for group affiliations)………………………………..……………………36 Figure 3.1. Scanning electron microscope images of Az-3 (left) and Az-1 (right) cells. Scale bar indicates 1 micron……………………………………………………………………..58 Figure 3.2. Average Absorbance of Strains Az-1, Az-2, and Az-3 at different Temperatures…..59 Figure 3.3. Average Absorbance of Strains 6a3T, PS, and SUA2T at different Temperatures…..59 Figure 3.4. Absorbance vs. Wavelength for Strains Az-1 and SUA2T. The dashed line denoted 235 nm, the maximum absorbance for the detection of crotonic acid……..…………...62 Figure 3.5. Absorbance vs. Wavelength during the measurement of crotonic acid as an indicator of PHB production by strains 6a3T, PS, Az-2, and Az-3. The dashed line denotes 235 nm, the maximum absorbance for the detection of crotonic acid…………………………...62

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ABSTRACT

In an effort to better understand the role that various microbes may play in toluene production, bacteria from a toluene-producing enrichment culture derived from contaminated groundwater at a Superfund site were cultivated and isolated on low nutrient solid media. A total of 14 solid medium formulations containing varying pH ranges, carbon sources, solidifying agents, and incubation gas headspaces were used to obtain 278 isolates in pure culture. Isolated bacteria, identified using partial 16S rRNA gene sequences, were most closely related with the genera Anoxybacillus, Azospira, Bacillus, Bradyrhizobium, Cellulosimicrobium, Micrococcus, and Propionicimonas.

Further attempts to target putative toluene-producing organisms made use of a screening approach involving a plate washing technique in conjunction with PCR amplification employing primers specifically designed to target 16S rRNA gene sequences unique to a subset of bacteria clustering within the phylum Acidobacteria. Experiments resulted in the recovery of an additional 56 bacterial isolates related to the genera Agrobacterium, Bacillus and Rhizobium.

Three isolates were selected for comparative testing to clarify their taxonomic positions.

Nearly complete 16S rRNA gene sequences determined for the three isolates were identical to one another and were most closely related to but clearly different from strains of the related species Azospira oryzae and Azospira restricta. Comparative testing revealed multiple of phenotypic differences between the new isolates and the reference strains. Collectively, data suggest that the new isolates represent a novel species.

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CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW 1.1. Introduction

It was recently reported that toluene appeared in groundwater [sometimes at concentrations far above the U.S. drinking water maximum contaminant level (MCL) of 1 mg/L] following the implementation of a bioremediation strategy aimed at anaerobic chlorinated solvent biodegradation at a Superfund site located north of Baton Rouge, Louisiana. Laboratory enrichment cultures demonstrated that toluene production was a biologically mediated process

(Moe et al., 2018). The microorganisms responsible for toluene production in the groundwater at this location have not yet been identified. Lack of understanding of the toluene producing organisms and conditions under which they thrive hinders informed decision making on how to avoid or minimize toluene production. Likewise, lack of knowledge regarding potential toluene consuming organisms that may also be present at the site hinders understanding of whether indigenous microbes may anaerobically biodegrade toluene near the area where it is produced.

The goal of research reported in this thesis was to isolate, identify, and characterize representatives from the microbial community that may contribute to toluene production or consumption. It is envisioned that a better understanding of the microbial populations will ultimately aid in decision making and improve remediation strategies at a Superfund site located near Baton Rouge, LA and other contamination sites around the world.

1.2. Literature Review

Industrial contaminants found in soil and groundwater have been prevalent since the rise of industrial production (Colten, 1991). At a global scale, halogenated organic compounds, specifically chlorinated alkanes and alkenes, are widespread environmental pollutants (De

Wildeman and Verstraete, 2003). These are primarily used in industry as solvents, paint

removers, degreasing agents, or intermediate compounds in other manufacturing processes (De

Wildeman and Verstraete, 2003). Due to large scale industrial production, which was estimated at about twenty-five million tons per year in the early 2000s for 1,1,1-trichloroethane and 1,2- dichloroethane alone, it is not surprising that these chemicals are frequently found in groundwater and soils (De Wildeman and Verstraete, 2003). In many environmental settings, these contaminants have relatively long half-lives (Vogel, 1987), and their toxicities can pose a threat to exposed humans and the surrounding environment (De Wildeman and Verstraete,

2003).

At the local level, groundwater at a Superfund site located near the Alsen community north of Baton Rouge, Louisiana is contaminated with a variety of chlorinated alkenes (e.g., tetrachloroethene, trichloroethene, trans-1,2-dichloroethene, cis-1,2-dichloroethene, and vinyl chloride) and chlorinated alkanes (e.g., 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2- dichloroethane and 1,2-dichloropropane) (USEPA, 2017). Contamination resulted from disposal of various chemical wastes in unlined earthen basins in the 1960s and 1970s, and these constituents were found to and pose a health risk if not mitigated (USEPA, 2017).

In-situ bioremediation has the potential to transform chlorinated solvents in contaminated groundwaters to non-toxic final products (e.g., ethene, ethane, and propene) due to anaerobic dehalogenation by some bacterial species (Maymó-Gatell et al., 1997). Several halorespiring anaerobes belonging to the phylum Chloroflexi have been reported in the literature. Among the best studied of these is Dehalococcoides mccartyi strain 195 (formerly informally referred to as

“Dehalococcoides ethenogenes” strain 195), the first bacterium demonstrated to completely dehalogenate tetrachloroethene (PCE) and trichloroethene (TCE) to ethene (Maymó-Gatell et al.,

1997; Fennell et al., 2004). Additional Dehalococoides mccartyi strains were also found to

transform vinyl chloride to ethene (Löffler et al., 2013). Also among the dechlorinating

Chloroflexi are representatives of the genus Dehalogenimonas. The type strains of three

Dehalogenimonas spp. (D. alkenigignens, D. formicexedens, and D. lykanthroporepellans) were isolated from groundwater at a south Louisiana Superfund site and were found to dehalogenate a variety of polychlorinated aliphatic alkanes from the same site (Moe et al., 2009; Bowman et al.,

2013; Key et al., 2017). Both Dehalococcoides and Dehalogenimonas are strict anaerobes, and compounds that are known to be utilized as electron acceptors are limited to halogenated organic compounds (Moe et al., 2009; Löffler et al., 2013). The compounds known to serve as electron donors for these bacterial genera also appear to be limited, with hydrogen (H2) used by

Dehalococcoides (Löffler et al., 2013) and H2 or formate used by Dehalogenimonas (Key et al.,

2017).

Because the chlorinated compounds serve as electron acceptors under anaerobic conditions, successful implementation of in situ bioremediation requires a supply of electron donor. One approach for providing electron donors for dechlorinating Chloroflexi is to inject a water-soluble, fermentable substrate, (e.g., molasses or sugars) into the subsurface (Moe et al., 2018).

Indigenous microbes use the molasses (or other compounds) and fermentatively produce H2 (and other compounds) in situ. The biologically produced hydrogen can then be used by the dechlorinating bacteria (Lee et al., 2004). Organisms such as those from the genus Clostridium have been found to produce hydrogen even in the presence of chlorinated solvents, putatively supporting dehalogenation through intra-species hydrogen transfer (Bowman, 2009).

At a Superfund site near the Alsen community in south Louisiana, agricultural feed grade cane molasses was repeatedly injected into groundwater in multiple areas in an effort to stimulate in situ reductive dechlorination (Moe et al., 2018). The approach was successful at stimulating

growth of dechlorinating Chloroflexi (Chen et al., 2014) and has proven successful in treating the target halogenated alkane and alkene contaminants (LDHH, 2014).

While the subsurface injection of agricultural feed grade molasses was effective in stimulating anaerobic reductive dechlorination, the aromatic hydrocarbon toluene was detected in groundwater sampled from a subset of the groundwater wells following molasses injections

(Moe et al., 2018). Toluene was not detected in the groundwater prior to molasses injection but in some cases reached levels far exceeding the federal drinking water maximum contaminant level (MCL) of 1 mg/L afterward. Benzene, ethylbenzene, and xylenes, common co- contaminants with toluene in areas impacted by gasoline, were below detection. Two independently-developed enrichment cultures from groundwater collected from different wells on different days consistently produced toluene in incubations supplied with phenylacetate or phenylalanine as precursors. It was concluded that toluene production is a biologically mediated process (Moe et al., 2018).

Biologically mediated toluene production in the environment has been reported previously, but there is uncertainty regarding the identities of the responsible organisms. The seasonal accumulation of toluene in the anoxic hypolimnion of a stratified lake in Germany led to the speculation that microbial processes could lead to toluene production with the aromatic amino acid phenylalanine as an essential precursor (Jüttner and Henatsch, 1986). A bacterium subsequently identified as Tolumonas auensis strain TA 4T was isolated from anoxic freshwater lake sediments and reportedly produced high concentrations (tens of mg/L) of toluene when provided with phenylalanine, phenylpyruvate, phenyllactate, or phenylacetate as precursors

(Fischer-Romero et al., 1996). Additional studies attempting to replicate toluene production by

Tolumonas auensis strain TA 4T and Clostriduium aerofoetidum, the only defined toluene-

producing isolates reported thus far, however, were unsuccessful (Zargar et al., 2016). While unsuccessful in reproducing toluene production by Tolumonas auensis TA 4T or Clostridium aerofoetidum, Zargar et al. (2016) was able to develop an enrichment culture derived from anaerobic sewage sludge that produced toluene from phenylacetate.

Although the genus Tolumonas was not found in the community analysis based on sequencing partial 16S rRNA genes, Beller et al. (2018) recently identified a toluene-producing phenylacetate decarboxylase (PhdB) enzyme and its cognate activating enzyme (PhdA) through a combination of metagenomics and in vitro confirmation of activity with recombinant enzymes

The genes encoding PhdB and PhdA were identified in a genome assembly that contained a partial 16S rRNA gene that allowed the unisolated bacterium that harbors the genes coding for toluene production to be assigned to the phylum Acidobacteria. Consequently, Beller et al.

(2018) refer to the bacterium harboring the phdA and phdB genes as Acidobacteria strain Tolsyn.

Based on 16S rRNA gene sequences, the closest previously cultured bacterium to the

Acidobacteria strain Tolsyn, with a sequence identity of only about 95%, is strain Ellin345

(Beller et al., 2018). Strain Ellin345, reported by Sait et al. (2002), was isolated from a soil core collected from a rotationally grazed pasture of perennial ryegrass and white clover at a research dairy in Australia. Ward et al. (2009) reported the genome sequence of the Ellin345 strain and informally proposed the name “Candidatus Koribacter versatalis” for the group that the strain represents.

The phylum Acidobacteria contains at least 26 major subgroups, a large fraction of which do not yet have cultivated representatives (Barnes et al., 2007). Members of subgroups 1, 3, 4, and 6 often dominate soil habitats while other subdivisions have been found to dominate cave mat communities, oceanic sediments, hot springs, or hydrothermal vents (Myers and King, 2016).

“Candidatus Koribacter versatalis” strain Ellin345 and the bacterium referred to by Beller et al.

(2018) as Acidobacteria strain Tolsyn falls within the group commonly referred to as subdivision

1. From a formal taxonomic perspective, they belong to the family Acidobacteriaceae (Foesel et al, 2016). Phylogenetically related subdivision 1 Acidobacteria based on a phylogenetic tree produced by Myers and King (2016) using 16S rRNA gene sequences and the maximum likelihood algorithm are presented in Table 1.1. Members of this group have generally been isolated under aerobic conditions on acidic media containing dilute concentrations of carbon sources (Kielak et al., 2016). Among media employed is VL55 (Sait et al., 2002) or variations of

VL55 medium solidified with gellan gum rather than agar or variations comprised of VL55 as a base but with various other carbon sources substituted for xylan (Joseph et al., 2003). This latter approach resulted in the isolation of multiple “Ellin345/WD217 group” bacteria, with a total of

29 of 350 randomly selected isolates clustering in Acidobacteria subdivision 1 (with

“Ellin345/WD217 group“ bacteria comprising 4 of the 29 Acidobacteria subdivision group 1 isolates) (Joseph et al., 2003).

Table 1.1. Phylogenetically related subdivision 1 Acidobacteria based on 16S rRNA gene sequences and maximum likelihood algorithm presented in Myers and King (2016). Phylogenetically related subgroup 1 Acidobacteria Reference Acidobacterium capsulatum strain JCM 7670T (Kishimoto et al., 1991) Acidipila rosea strain AP8T (Okamura et al., 2011) Acidobacterium ailaaui strain PMMR2T (Myers and King, 2016) ‘Koribacter versitallis’ strain Ellin345 (Ward et al., 2009) ‘Silvibacterium bohemicum’ strain S15T (Lladó et al., 2016)

Subdivision 1 Acidobacteria are generally slow growing and poorly represented in many bacterial communities compared to other numerically dominant species (Sait, 2008). Long incubation times (up to 168 days) may be required to obtain visible colonies on the solid media

(Campanharo et al., 2016). They may also require interactions with other microbes for growth or

depend on specific growth conditions to compete (Sait, 2008). A solid medium formulation referred to as PSYA 5 was used to cultivate and enumerate various strains in the phylum

Acidobacteria using varying pH, organic carbon sources, and solidifying agents (Campanharo et al., 2016). It was found that cell growth was most significant at a pH of 5.5 using sucrose as a carbon source and an incubation temperature of 30˚C. The growth of three strains of subdivision

1 Acidobacteria (WH120, WH15, and 5B5) was achieved under aerobic conditions (Campanharo et al., 2016). Alternatively, Sait (2008) isolated six strains phylogenetically affiliated with the phylum Acidobacteria on VL55 media. Ellin345, the most closely related organism to

Acidobacteria strain Tolsyn based on 16S rRNA gene sequences, was isolated on VL55 with washed agar as the solidifying agent and 0.05% (w/v) xylan as a carbon source. Ellin345 formed

1-2 mm white, circular colonies with an entire edge and a glistening surface after 12 weeks incubation at 25°C. It also grew well from pH 4 to 6 at 25°C but did not grow with 5 g/L NaCl.

When incubated in elevated CO2, there was no apparent change in biomass formation (Sait,

2008). In an alternate study, the frequency of Acidobacteria detected on solid media increased to

22.5% when incubated in 5% CO2. The frequency of Acidobacteria detection also increased from 12% to 25% when the oxygen concentration was decreased from 21% to 2% (Stevenson et al., 2004).

Group-specific PCR primers have been established to target Acidobacteria and its subgroups.

Lee and Cho (2011) designed the primer ACIDO which showed an increase in coverage for the entire phylum Acidobacteria when compared to primer 31F, an Acidobacteria-targeted primer designed by Barnes et al. (1999) with only 45.5% of sequences belonging to Acidobacteria having complementary binding sites. Eighty-three percent of sequences had complementary binding sites to primer ACIDO, and it was able to detect sequences from all six subgroups (Lee

and Cho, 2011). Also, primers S1, S2, and S3 were designed to target Acidobacteria subgroups

1, 2 and 3, respectively. These primers showed high specificity in studies with a large number of sequences analyzed (Lee and Cho, 2011). However, Lee and Cho (2011) found that Primer S1 showed lower specificity for targeting the Acidobacteria subgroup 1 than what was expected from in silico analysis.

In additional experiments conducted in the Moe laboratory using the toluene-producing enrichment cultures derived from the Louisiana Superfund site, 16S rRNA gene libraries constructed using community DNA extracts of various generations of the toluene producing cultures were found to contain a sizable percentage of sequences (up to 7%) closely related to

Acidobacteria strain Tolsyn (unpublished data). 16S rRNA gene sequences most closely related to other bacterial genera including Propionicimonas and Azospira were found at substantial abundances as well (e.g., 0.78% and 72.8%, respectively) (unpublished data). The presence of these microbial groups in the enrichment cultures that accumulated high toluene concentrations raises the question about whether they may play an important role in the production or consumptions of toluene. Because toluene accumulation in the groundwater likely depends on both the local rate of toluene production and the rate of local consumption (if it is non-zero), better understanding the role of various microbial components in toluene production and consumption both have the potential to aid in improving contaminant fate and transport modelling and in improving decision-making in the remediation field.

1.3. Research Objectives

The overall objective of the research described in this thesis was to isolate, identify, and characterize bacteria from a toluene-producing enrichment culture derived from a Louisiana

Superfund Site, with the goal of identifying community components that may contribute to toluene transformation or otherwise impact remediation efforts.

1.4. Thesis Organization

Following the introduction and literature review presented here in Chapter 1, Chapter 2 describes bacterial cultivation and isolation experiments on various solid media. Additional experiments including Acidobacteria-specific primer design and implementation as well as toluene production experiments using individual isolates are also described in Chapter 2. Chapter

3 describes the further characterization and comparative testing of three bacterial strains isolated in experiments described in Chapter 2. Overall conclusions and recommendations for future research are presented in Chapter 4. References cited throughout the thesis can be found at the end.

CHAPTER 2. CULTIVATION AND ISOLATION OF BACTERIA DERIVED FROM A TOLUENE-PRODUCING ENRICHMENT CULTURE ON SOLID MEDIA

2.1. Introduction

This chapter describes experiments aimed at the isolation and identification of bacteria from a toluene-producing enrichment culture. The general process flow for experimentation described in this chapter is shown in the flow chart to follow.

2.2. Toluene-Producing Enrichment Cultures

The inoculum used to cultivate, isolate, and identity bacteria from a previously described

Superfund Site in north Baton Rouge (Moe et al., 2018) originated from three different generations of a sugar-free, toluene-producing, enrichment culture. The parent enrichment culture was derived from groundwater sampled from injection well ID number SP022 at the site on April 24, 2017 at a time of 222 days after the fourth injection of molasses amended groundwater. The well location and general well construction details are summarized in Table

2.1. below.

Table 2.1. Groundwater well location from which the SR enrichment culture originated [data from Moe et al. (2018)]

Class-V Injection DTZ Well No. SP022 Well serial number 974204 Date installed 1-23-12 Latitude (NAD 83) 30° 35’ 42.5489” Longitude (NAD 83) 91° 14’ 42.9001” Depth (feet bgsa) 108 feet Screened interval (feet bgsa) 55-105 feet Top of casing elevation (NAVD 88) 76.28 feet a bgs = below ground surface Contaminant concentration (Table 2.2.) and geochemical parameters (Table 2.3.) for the groundwater at the time of collection for establishment of the enrichment culture are summarized below.

Contaminated Groundwater

Toluene-producing SR enrichment culture with glucose

Dilution-to-extinction

-4 -7 experiments in sugar- 10 -6 10 10-5 10 free medium

Diluted, sugar-free, toluene- producing SR enrichment culture 10-7

18 variants of VL55 media (VL-variants) and 2 variants of solid PA2D media

278 bacterial isolates and concentrated cell pellets from plate wash analysis

DNA extraction of isolates and plate washed colonies

PCR amplification using universal 16S rRNA gene primers or Acidobacteria-specific primers

DNA sequencing and tentative identification

Table 2.2. Volatile organic compound (VOC) concentrations measured in groundwater collected for establishment of enrichment cultures from well SP022 on April 24, 2017 [222 days after the fourth molasses injection, data from Moe et al. (2018)].

Concentration Analyte* Well SP022 Benzene (µg/L) <0.200 1,2-Dichloroethane (µg/L) <0.200 1,2-Dichloropropane (µg/L) <0.200 cis-1,2-Dichloroethene (µg/L) <0.200 trans-1,2-Dichloroethene (µg/L) <0.200 Ethylbenzene (µg/L) <0.200 1,1,2,2-Tetrachloroethane (µg/L) <0.200 Tetrachloroethene (µg/L) <0.200 Toluene (µg/L) 7,670 1,1,2-Trichloroethane (µg/L) <0.200 Trichloroethene (µg/L) <0.200 Cholorbenzene (µg/L) <0.200 Vinyl chloride (µg/L) 10.8J *Analyzed using US EPA Method 8260B J Estimated value (result between the method detection limit and limit of quantification) Table 2.3. Geochemical parameters measured in groundwater collected for establishment of enrichment cultures from well SP022 on April 24, 2017 [data from Moe et al. (2018)].

Concentration Analyte Well SP022 Ethene (µg/L) <0.150 Ethane (µg/L) 0.418J Methane (µg/L) 10,200 Total organic carbon (mg/L) 154 Chloride (mg/L) 125 Nitrate (mg/L-N) <0.008 Nitrite (mg/L-N) 0.032 Sulfate (mg/L) 1.64J Sulfide (mg/L) 0.828 Ferrous iron (mg/L) 3.27 pH (SU) 7.31 Total Alkalinity (mg/L as CaCO3) 2,110 ORP (mV) -280 Temperature (°C) 20.3 Conductivity (µmhos/cm) 4,185 Turbidity (NTU) 19.2

J Estimated value (result between the method detection limit and limit of quantitation)

The initial enrichment culture, designated as SR, was established by inoculating groundwater (4% v/v) into modified TP medium as described by Zargar et al. (2016) but with phenylacetic acid added to a final concentration of 2.47 mM (339 mg/L), pH 7.6. The headspace gas was comprised of 95% N2, 5% CO2. Incubation was at ambient laboratory temperature (21±2

°C), close to the groundwater temperature (see Table 2.2.), in the dark without mixing. The culture was serially transferred (0.1-10%, v/v) at 2-8 week intervals in the same medium incubated at the same temperature (Moe et al., 2018). In the modified TP medium, which contained 1 g/L glucose, the SR enrichment culture produced toluene when supplied with phenylacetic acid, phenylalanine, phenyllactate, or phenylpyruvate. Toluene production could be sustained over multiple serial transfers (at least 9) and could be recovered from samples diluted to at least 10-7. Experiments conducted using 13C labelled compounds (phenylacetic acid-2-13C and L-phenylalanine-3-13C) resulted in production of toluene-α-13C, confirming that toluene was synthesized from these precursors by two independently developed enrichment cultures (Moe et al., 2018).

In subsequent experiments (unpublished data), the SR culture was sequentially transferred in modified TP medium containing 350 mg/L phenylacetic acid but without glucose addition. Toluene production could be sustained over at least 10 consecutive transfers in the sugar-free medium and could be recovered from relatively deep dilution (10-7) and after brief oxygen exposure (aeration for one hour) (unpublished data from S. J. Reynolds). These subsequently transferred enrichment cultures, specifically from the second, sixth, and eighth generations of dilution-to-extinction experiments in sugar free medium, were used for cultivation attempts on solid media because the active cultures were capable of producing high concentrations of toluene (>100 mg/L) within as little as 30 days and 16S rRNA gene libraries

constructed using Nextgen sequencing (Illumina) revealed that a handful of phyla dominated the sugar-free cultures (unpublished data). For example, a toluene producing 10-7 dilution from the third generation in sugar-free medium was found to include <30 operational taxonomic units

(OTUs) with sequences clustering with Azospira (73%) and “Koribacter” (7%) comprising a large fraction of the total sequences (unpublished data). As described in the following subsections, cultivation techniques employing solid media were applied.

2.2. Medium Preparation and Inoculation

Eighteen variants of low nutrient solid media (VL-variants) and two variants of solid

PA2D media were used to propagate organisms from enrichment cultures. Low nutrient solid media was used as an attempt to target potential toluene producers. VL55 media showed success in supporting growth for many organisms from the phylum Acidobacteria as well as other rarely cultured bacteria (Sait, 2008). Solid PA2D media was used to mimic the conditions of the toluene-producing liquid inocula. Variations of the VL55 media were prepared following the general procedure from the DSMZ (DSMZ medium 1266, Medium VL55), and variations of

“PA2D medium” (described below) were prepared following the method of Zargar et al. (2016).

All variations are summarized in Table 2.4. below.

Solid VL55 media was prepared by combining the following constituents (per liter): 2-

Morpholinothanesulfonic acid (MES), 1.95 g; 20 mM MgSO4·7H2O, 10 mL; 30 mM CaCl2·2

H2O, 10 mL; 20 mM (NH4)2HPO4, 10 mL; selenite-tungstate solution, 1 mL; trace element solution, 1 mL; and distilled water (see section 2.3 for composition of stock solutions). If xylan was used as an alternative carbon source, it was added to achieve a concentration of 2 mM prior to sterilization. The media was then adjusted to the desired pH, ranging from 5.5 to 7.2 using 200 mM NaOH/100 mM KOH solution and autoclaved for sterilization. Vitamin solution and 0.2 M

glucose (when added) were added to the media from filter-sterilized stocks after autoclaving.

Either washed agar (BD Bacto Agar) or Gelzan CM (Sigma) was used to solidify the media with preparation as described below.

Washed agar was prepared by combining 16.5 g/L BD Bacto Agar with deionized water

(18Ω, Milli-Q). The suspension was stirred for 5 minutes using a magnetic stir bar and stir plate, then allowed to settle for 30 minutes. The supernatant was decanted, and the process was repeated 5 times. The washed agar was resuspended in deionized water to a final concentration of 3%, m/v and autoclaved. After autoclaving and cooling to approximately 50°C, the washed agar was combined 1:1 (v/v) with the VL55 liquid media prior to distributing into petri dishes.

When the alternative solidification agent Gelzan CM was employed, it was added to the VL55 medium prior to autoclaving at a concentration of 8 g/L. Liquid media were prepared in a similar manner but excluding the solidifying agents.

“PA2D medium” was prepared following the method described in Zargar et al (2016) for modified TP medium except that phenylacetic acid was supplied at a final concentration of 350 mg/L. The following constituents were added to a serum bottle (per liter): KH2PO4, 0.25 g;

NH4Cl, 0.34 g; KCl, 0.34 g; Sodium HEPES, 4.69 g; Glucose, 1 g; MgCl2·6H2O, 1 g;

MgSO4·7H2O, 0.1 g; CaCl2·2H2O, 0.125 g; phenylacetic acid, 0.35 g; yeast extract solution, 1%, v/v ; and trace element solution, 0.05%, v/v. Deionized water that had been boiled for 20 minutes was added to the serum bottle containing the previously described constituents. If Gelzan CM was used, it was added at a concentration of 8 g/L prior to autoclaving. The bottle was capped with a butyl rubber stopper and sealed with an aluminum crimp cap. A hypodermic needle was then inserted through the butyl stopper and used as a gas vent. A 6 inch stainless steel needle connected to a gas cylinder containing 95% N2: 5% CO2 was also inserted through the butyl

rubber stopper. The media was purged with the anaerobic gas for 5 minutes and then autoclaved.

Once sterile and cool, 0.028%, v/v vitamin B12 stock solution was added to the media. The liquid media used for preparing dilution was prepared in a similar way except excluding the solidifying agent. The media bottle was opened and combined with washed agar (when washed agar was used), and dispensed into disposal, sterile polystyrene petri dishes (VWR).

Variants of the growth media (Table 2.4.) included various pH levels, the use of xylan or glucose as a carbon source, the use of washed agar or Gelzan as a solidifying agent, and the addition or absence of 2%, v/v NaCl, 1 g/L ampicillin, or 0.1 g/L vancomycin. The inoculated plates were also incubated in varying gas headspace conditions of aerobic (incubation in ambient air), 5% CO2, and anaerobic conditions. The 5% CO2 and anaerobic headspace conditions were achieved using BD GasPak™ CO2 generator sachets or anaerobic sachets with the indicator placed inside BD GasPak™ EZ containers or pouch systems.

Table 2.4. Growth Media used to Isolate Colonies from the Toluene-Producing Enrichment Cultures

Label Carbon pH Solidifying Incubation Source Agent Conditions

MM 5.5 Glucose 5.5 Washed agar Aerobic VL55* 5.5 Glucose 5.5 Gelzan Aerobic VL55 5.5 GX Xylan 5.5 Gelzan Aerobic VL55 5.5 AX Xylan 5.5 Washed Agar Aerobic VL55 5.5 Glucose 5.5 Gelzan Aerobic, 5% CO2 OVL55 5.5 Glucose 5.5 Washed Agar Aerobic VL55 5.5 with 2%, v/v Glucose 5.5 Washed Agar 5% CO2 NaCl VL55 6, 0.1g/L Glucose 6.0 Gelzan 5% CO2 vancomycin VL55 6, 1g/L ampicillin Glucose 6.0 Gelzan 5% CO2

VL55 6 Glucose 6.0 Gelzan Aerobic, 5% CO2 VL55 6.5 Glucose 6.5 Gelzan Aerobic, 5% CO2, Anaerobic VL55 6.5 GX Xylan 6.5 Gelzan Aerobic, Anaerobic OVL55 6.5 Glucose 6.5 Washed Agar Aerobic VL55 6.5 + 2%, v/v NaCl Glucose 6.5 Washed Agar 5% CO2 + 0.1 g/L vancomycin + 1 g/L ampicillin VL55 7 Glucose 7.0 Gelzan Aerobic, 5% CO2 ZPA2D A Glucose 7.0 Washed Agar 5% CO2 ZPA2D G Glucose 7.0 Gelzan 5% CO2, Anaerobic MM 7 Glucose 7.0 Washed agar Aerobic VL55* 7 Glucose 7.0 Gelzan Aerobic VL55 7.2 AX Xylan 7.2 Washed Agar Aerobic

Working on a clean bench, dilutions of the inoculum were made in medium identical in composition to the solid media but without solidifying agent. Using 0.5 mL of the parent culture,

10-fold dilutions out to 10-12 of the parent inoculum were prepared. Each dilution was vortexed for 15 seconds at setting 3 on a VortexGenie 2 (Scientific Industries, Inc.) prior to making the following dilution. Aliquot volumes of either 0.1 or 0.2 mL from each liquid dilution were pipetted on to the plates and spread with a sterile plastic spreader. Plates were prepared in triplicate or quadruplicate for each dilution. To prevent drying during a long incubation time,

plates with ambient air in the gas headspace were wrapped in parafilm and stored in a plastic sleeve. Inoculated plates were incubated at 25°C and visually observed once every two weeks for growth. Plates incubated for a minimum of 21 days.

2.3. Stock Solution Preparation

2.3.1. VL55 Stock Solutions

VL55 Vitamin solution was prepared by combining the following constituents (per liter): vitamin B12, 17 mg; 4-aminobenzoate, 13 mg; biotin, 3 mg; nicotinic acid, 33 mg; hemicalcium

D-(+)- pantothenate, 17 mg; pyridoxamine-HCl, 50 mg; thiamine-HCl·2H2O, 33 mg; D,L-6,8- thioctic acid, 10 mg; riboflavin, 10 mg; folic acid, 4 mg; and deionized water. The solution was then filter sterilized using a 0.2 µm sterile Posidyne® Membrane syringe filter (PALL Life

Sciences) into a sterile serum bottle and sealed with a butyl rubber stopper and aluminum crimp cap. The bottle was covered with aluminum foil to prevent light exposure and stored in a refrigerator.

SL-10, trace element solution used for the VL55 media was prepared by dissolving

FeCl2·4H2O, 1.5 g; in 7.7 M (25%) HCl, 10 mL; then diluting into 0.99 L deionized water to make a 1 L solution. The following salts were dissolved into the 1 L solution: ZnCl2, 70 mg;

MnCl2·4H2O, 100 mg; H3BO3, 6 mg; CoCl2·6H2O, 190 mg; CuCl2·2H2O, 2 mg; NiCl2·6H2O, 24 mg; and Na2MoO2·2H2O, 36 mg.

The VL55 selenite-tungstate solution contained NaOH, 500 mg; Na2SeO3·5H2O, 3 mg; and Na2WO4·2H2O, 4 mg per liter with deionized water. It was autoclaved in 50 mL aliquots in glass screw cap bottles.

2.3.2. PA2D Stock Solutions

Vitamin B12 stock solution used for PA2D medium was prepared by combining vitamin

B12, 0.1 g/L; with deionized water. The solution was filter sterilized into a sterile glass serum bottle using a 0.2 µm sterile Posidyne® Membrane syringe filter (PALL Life Sciences) and sealed with a butyl rubber stopper and an aluminum crimp cap. The headspace was purged with a sterile source of O2-free gas consisting of 95% N2: 5% CO2 for 5 minutes. The solution was wrapped in aluminum foil to prevent light exposure and stored at a cold temperature.

PA2D yeast extract solution was made by adding 1 g/L BD Bacto Yeast Extract to deionized water that was boiled for 20 minutes. The bottle was sealed with a butyl rubber stopper and aluminum crimp cap. The solution was purged with an anaerobic gas supply consisting of

95% N2: 5% CO2 for 5 minutes before autoclaving.

The trace element solution used for PA2D media was prepared by combining the following constituents with deionized water (per liter): 7.7N (25%) HCl, 1.25%, v/v;

FeSO4·7H2O, 2.1 g; MnCl2·4H2O, 100 mg; CoCl2·6H2O, 190 mg; ZnCl2, 70 mg; NiCl2, 13 mg;

CuCl2·2H2O, 2 mg; Na2MoO4·2H2O, 36 mg; H3BO3, 6 mg. The gas headspace of the solution was purged with anaerobic gas (95% N2: 5% CO2) for 5 minutes, and the solution was autoclaved to sterilize.

2.3.3. Additional Stock Solutions

Antibiotic stock solutions were used in some media variations. A 100× ampicillin stock solution was prepared by combining 100 g/L ampicillin sodium salt (Sigma-Aldrich) with deionized water. Similarly, a 100× vancomycin stock solution was prepared by combining 10 g/L vancomycin hydrochloride from Streptomyces orientalis (Sigma) with deionized water. The

solutions were filter sterilized using a 0.2 µm sterile Posidyne® Membrane syringe filter (PALL

Life Sciences). The gas headspaces were purged for 5 minutes using filter sterilized anaerobic gas (95% N2: 5% CO2). The sterile stocks were then added to the sterile growth media at a concentration of 1%, v/v.

2.4. Experimental Design for Isolation and Identification of Bacteria

2.4.1. DNA Extraction and Plate Wash PCR (PWPCR)

Once the plates had visible colonies, well-isolated colonies were individually selected and streaked onto a new plate of the same composition using sterile 1 µL disposable inoculating loops. Colonies were sequentially transferred two to three times onto the same medium until morphologically homogenous colonies were visually observed.

For DNA extraction, isolated colonies were picked from the purified streak plates using sterile 10 µL pipette tips. The tip of the pipette, which contained most or all of the picked colony, was clipped off (using sterile stainless steel scissors) into to a sterile 1.5 mL microcentrifuge tube. DNA was then extracted using a QIAamp® DNA Micro Kit (Qiagen) following the manufacturer’s protocol for isolation of genomic DNA from tissues. In this procedure, immediately after collecting colonies as described above, 180 µL Buffer ATL and 20 µL proteinase K were added to the microcentrifuge tube and mixed by pulse-vortexing for 15 seconds. The samples were placed in a 56°C water bath overnight for cell lysis. Following lysis,

200 µL Buffer AL was added, and the sample was mixed by vortexing for 15 seconds. Next, 200

µL 200 proof ethanol (molecular grade) was added, mixed by vortexing, and incubated for 5 minutes at room temperature. The entire lysate was transferred to a QIAamp MiniElute column

(in a clean 2 mL collection tube). It was centrifuged at 6,000 × g for 1 minute. The column was then placed in a clean 2 mL collection tube while the tube with the flow-through was discarded.

In the same MiniElute column, 500 µL Buffer AW1 was added carefully without wetting the rim, centrifuged at 6,000 × g rpm for 1 minute, and placed in a new collection tube. This was repeated but with 500 µL Buffer AW2. The column was placed in a clean 2 mL collection tube and centrifuged at full speed (18,900 × g) for 4 minutes to dry the membrane. The flow-through was discarded, and the MiniElute column was placed in a clean 1.5 mL microcentrifuge tube. To elute the DNA, 20 µL Buffer AE was added to the center of the membrane and the tube incubated at room temperature for 5 minutes. It was centrifuged at full speed (18,900 × g) for 2 minutes. The eluted DNA was stored at -80°C until future use. In cases where low DNA concentrations were expected (for small (< 0.2 mm) colonies), carrier RNA was used to increase the yield as described by the kit’s instructions. Unless otherwise stated, pulse-vortexing was performed at setting 5 on a Vortex Genie 2 (Scientific Industries). The quantity and quality of the extracted DNA were analyzed spectrophotometrically using a Nanodrop 2000 (Thermo

Scientific). The concentration, A260, A280, 260/280, and 260/230 were recorded for a 2 µL sample.

Plates incubated in 5% CO2 or anaerobic conditions were not used for individual colony isolation and instead were analyzed via Plate Wash PCR (PWPCR) to screen for potential toluene-producing bacteria following a procedure similar to that described by Stevenson et al.

(2004). After four weeks of incubation, one representative plate from a dilution series showing abundant growth (typically one replicate from the most concentrated plates) was selected to be washed and DNA extracted. The plate was flooded with 3 mL sterile liquid media with the same composition as the solidified medium of the plate (but without solidifying agent). Using a sterile plastic spreader, colonies were dislodged from the surface and suspended into the liquid without breaking the surface of the agar or Gelzan. Approximately 1.5 mL of the liquid was pipetted into

a sterile 2 mL microcentrifuge tube and centrifuged for 1 minute at 4,500 × g. Supernatant was decanted, and then remaining liquid on the plate was pipetted into the same microcentrifuge tube.

The sample was centrifuged a second time, and then supernatant was decanted. The concentrated cell pellet was then used for DNA extraction following the protocol described above using the

QIAamp® DNA Micro Kit (Qiagen). This process was repeated for plates that incubated for an additional four weeks (eight weeks total).

2.4.2. PCR Amplification and Sequencing using Universal Bacterial Primers

DNA extracted from isolates was used as a template in PCR reaction using universal 16S rRNA gene primers 27f YM (Frank, 2008) and 1492r (Lane, 1991) (Table 2.5.). PCR was performed using a Veriti Thermal Cycler (Applied Biosystems) and a Phusion High-Fidelity

PCR kit (New England Biolabs, Inc). For each 25 µL reaction, the following components were combined on ice: Phusion DNA polymerase (0.25 µL, 0.5 U), 5× Phusion HF Buffer (5 µL), 10

µM Forward Primer (1.25 µL), 10 µM Reverse Primer (1.25 µL), 10 µM dNTPs (0.5 µL), template DNA (2 µL), and nuclease-free water (14.75 µL). Samples were gently mixed and briefly spun down in a microcentrifuge (10 seconds) before placement in the thermal cycler. The thermal program consisted of initial denaturation at 98°C for 5 minutes followed by 40 cycles of denaturation for 10 seconds at 98°C, 30 seconds annealing at 55°C, and 45 seconds extension at

72°C followed by a final extension at 72°C for 10 minutes. PCR products were electrophoresed on a 2%, w/v agarose gel for 45 minutes at 72 to 74 V. Imaging was performed using a BioRad

UV gel imaging system used in conjunction with Quantity One 4.4.0 software.

Table 2.5. Universal bacterial Primers Used for Isolates

Primer Sequence (5’-3’) Annealing Reference Name Temperature with Phusion (°C)

27f YM AGAGTTTGATYMTGGCTCAG 55 Frank (2008)

1492r TACGGYTACCTTGTTACGACTT Lane (1991)

PCR amplicons produced using universal primers 27f YM and 1492r were sequenced via

Sanger sequencing performed on ABI 3730xl DNA Sequencer at Eton Bioscience, Inc.

(https://www.etonbio.com). Resulting fasta files from sequenced isolates were compared against the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm

(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LIN

K_LOC=blasthome) to obtain an initial tentative identification. Partial sequences from sequenced isolates were assembled using Cap3 Sequence Assembly Program

(http://doua.prabi.fr/software/cap3) and then compared against NCBI database as well as

EZBioCloud 16S-based ID database (https://www.ezbiocloud.net/identify) to group related isolates and determine the closest related cultured and uncultured representatives.

2.4.3. Acidobacteria-specific Primer Design, PCR Amplification, and Sequencing

In addition to amplification using universal primers 27f YM and 1492r (Table 2.5.), community DNA isolated via plate washing was used as template in PCR reactions using previously described and newly developed primers (Tables 2.6. and 2.7.) intended to uniquely target group 1 of the phylum Acidobacteria.

Acidobacteria-specific primers developed in this study were designed using partial 16S rRNA gene sequences available from Nextgen sequencing of earlier generations of the toluene- 23

producing SR enrichment culture (unpublished data) and the closest related microorganisms reported in the public databases (e.g. Acidobacteria strain Tolsyn from Beller et al. 2018). The

BioEdit v.7.2.5 Sequence Alignment Editor (Hall, 1999) was used to manually align and mask sequences prior to primer refinement using Primer3Plus as further described below. 16S rRNA gene sequences from Acidobacteria strain Tolsyn (JGI2065J20421 10002126), other uncultured

Acidobacteria clones closely related (>98.0% identity) to strain Tolsyn (Genbank accession numbers HQ598817, HQ010168, AB364789, GQ402804, JF309170, HQ597146, JF309192),

Candidatus Koribacter versitalis Ellin 345 (NC_008009.1 :c5261562-5260062), isolated

Azospira and Propionicimonas strains determined from the present study (see Appendix A), related type strains within these genera (Azospira oryzae 6a3T (NR_024852.1), Azospira restricta

SUA2T (DQ974114), Propionicimonas ferrireducens Y1A-10-1T (MG775313), Propionicimonas paludicola WdT,(NR_104769)), and non-target bacteria with relatives present in past generations of the SR cultures, specifically from the genera Parabacteroides (NR_109439), Sulfuropirillum

(AB246781), Proteiniclasticum (NR_115875), and Chloracidobacterium (AB862122) were manually aligned in BioEdit. Non-target bacteria falling within Acidobacteria subgroup 1 but not closely related to strain Tolsyn or Candidatus Koribacter versitalis Ellin345 were also included in the alignment. These included Bryobacter aggregatus MPL3T (AM162405), Holophaga foetida TMBS4T (NR_036891), Geothrix fermentans HradG1 (HF559174, HF559175,

HF559181), Edaphobacter modestus Jbg-1T (DQ528760.2), Edaphobacter aggregans Wbg-1T

(DQ528761), Granulicella tundricola MP5ACTX9T (CP002480.1:c3732212-3730725),

Granulicella pectinivorans DSM 21001T (FOZL01000001.1:c4438537-4437035) Granulicella mallensis MP5ACTX8T (CP003130.1:691197-692684), and Bryocella elongata SN10T

(FR666706). Conserved regions across the sequences were identified and excluded from

potential primer design to prevent non-specific amplification. Candidate regions conserved between strain Tolsyn and related bacteria from the Superfund site groundwater and Superfund site enrichment cultures but not in distantly related groups were manually selected for primer design.

Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), with default parameters, was used to refine potential primers. The Ribosomal Database Project (RDP)

Probe Match tool (https://rdp.cme.msu.edu/probematch/search.jsp) was used to further evaluate specificity to the Candidatus Koribacter group within the phylum Acidobacteria. Five primers combined into three primer sets (hereafter referred to as “Tol1”, “Tol2”, and “Tol3”) were ultimately selected for further experimental testing (Tables 2.6. and 2.7.). The manually selected primers were assessed for potential dimerization using the Fisher Scientific Primer Analysis tool

(https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular- biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific- web-tools/multiple-primer-analyzer.html) and for potential secondary structures (e.g. hairpin structures) using Oligoevaluator (http://www.oligoevaluator.com/OligoCalcServlet).

Table 2.6. Universal and “Koribacter”-specific Primer Sets Used for Plate Wash PCR

Primer Set Primer Sequence (5’-3) Annealing Reference Name Name Temperature with Phusion (°C) Universal 27f YM AGAGTTTGATYMTGGCTCAG 55 Frank (2008) 1492r TACGGYTACCTTGTTACGACTT Lane (1991) Tol1 Tol1f AGAAACTGCCGTGCTTGAGT 65 This study Tol1r ACAGCAGGATTGGGTACCTG This study Tol2 Tol2f CAGGTACCCAATCCTGCTGT 65 This study Tol2r GGGCAGTTTCGCCAGAGT This study Tol3 Tol3f AAACTGCCGTGCTTGAGTAT 63 This study Tol2r GGGCAGTTTCGCCAGAGT This study

Table 2.7. Expected Amplicon Size from Primer Sets

Primer Set Amplicon Size Universal ~1465 Tol1 218 Tol2 329 Tol3 525

Additional phylum- and group-specific primers used are shown below in Table 2.8.

Primers S1, ACIDO, 31F, and primer A were used for initial plate wash PCR tests; however, primer sets Tol1, Tol2, and Tol3 were designed due to non-specific amplification or no amplification as further described in the results section. If these primers successfully amplified the toluene-producing organism(s) in the positive control (template DNA from an SR toluene- producing enrichment culture), they were used for amplification of plate washed samples.

Table 2.8. Additional Primers used for Acidobacteria-targeted PCR Primer Set Primer Sequence (5’-3’) Reference Phylum-specific ACIDO GCTCAGAATSAACGCTGG Lee and Cho (2011) 1492r TACGGYTACCTTGTTACGA Lane (1991) CTT Group-specific S1 CGCACGGMCACACTGAACT Lee and Cho (2011) 1385r CGGTGTGTRCAAGGCCC Rainey (1996) Phylum-specific 31F GATCCTGGCTCAGAATC Barnes et al. (1999) 1385r CGGTGTGTRCAAGGCCC Rainey (1996) Group-specific Primer GCCTGAGAGGGCRC Barnes et al. (1999) A 1385r CGGTGTGTRCAAGGCCC Rainey (1996)

Temperature programs for PCR reactions employing the various primer sets are presented in Table 2.9. PCR was performed using a Veriti Thermal Cycler, and samples were prepared with a Phusion High-Fidelity PCR kit by New England Biolabs, Inc (for primer sets Tol1, Tol2, and Tol3) or with AmpliTaq Gold™ DNA Polymerase kit by Applied Biosystems (for primer sets ACIDO-1492r, Primer A- 1385r, and S1-1385f). Temperature programs for PCR reactions using Phusion High-Fidelity PCR kit were similar to the protocol described in section 2.4.2

except with 20 seconds of annealing at 65°C for primer sets Tol1 and Tol2 and 20 seconds of annealing at 63°C for primer set Tol3.

Temperature programs for reactions using Amplitaq Gold™ Polymerase kit were prepared following the manufacturer’s protocol as described below. For each 25 µL reaction, the following components were combined on ice: AmpliTaq Gold DNA polymerase (0.125 µL,

0.625 U), 10× PCR Buffer I (2.5 µL), 10 mM dNTP Mix (0.5 µL), 10 µM Forward Primer (0.5

µL), 10 µM Reverse Primer (0.5 µL), template DNA (2 µL), and nuclease-free water (18.875

µL). Samples were gently mixed and briefly spun down in a microcentrifuge (10 seconds) before placement in the thermal cycler. The thermal programs consisted of initial denaturation at 95°C for 5 minutes from primer set ACIDO-1492r or 94°C for 2 minutes for primer sets Primer A-

1385r and S1-1385r followed by 30 cycles of denaturation for 60 seconds at 95°C (for primer set

ACIDO-1492r) or 94°C (for primer sets Primer A-1385r and S1-1385r), 60 seconds (for ACIDO-

1492r) or 30 seconds (for Primer A-1385r and S1-1385r) of annealing (annealing temperatures presented below in Table 2.9.), and 120 seconds (for ACIDO-1492r) or 60 seconds (for Primer

A-1385r and S1-1385r) extension at 72°C followed by a final extension at 72°C for 20 minutes

(for ACIDO-1492r) or 5 minutes (for Primer A-1385r and S1-1385r). Temperature programs for primer sets are summarized in Figure 2.9. below. PCR products were electrophoresed on a 2%, w/v agarose gel for 45 minutes at 72 to 74 V. Imaging was performed on a BioRad UV gel imaging system used in conjunction with Quantity One 4.4.0 software.

Table 2.9. Thermal Program for all Primer Sets

Primer # Initial Denaturation Annealing Extension Final Set Cycles Denaturation Extension T3 t4 T t T t T t T t (°C) (min) (°C) (sec) (°C) (sec) (°C) (sec) (°C) (min) Universal1 40 98 5 98 10 55 30 72 45 72 10 ACIDO- 30 95 5 95 60 58 60 72 120 72 20 1492r2 Primer A- 30 94 2 94 60 57 30 72 60 72 5 1385r2 S1-1385r2 30 94 2 94 60 57 30 72 60 72 5 Tol11 35 98 2 98 10 65 20 72 15 72 5 Tol21 35 98 2 98 10 65 20 72 15 72 5 Tol31 35 98 2 98 10 63 20 72 15 72 5 1 PCR performed with Phusion DNA polymerase 2 PCR performed with AmpliTaq Gold DNA polymerase 3 “T” represents the temperature at the specified step 4 “t” represents the time at the specified step

Confirmed PCR products using group-specific, phylum-specific, or “Koribacter”-specific primers were sent to Eton Bioscience, Inc. (https://www.etonbio.com/) for sequencing. Sanger sequencing was performed on ABI 3730xl DNA Sequencers. Resulting fasta files (partial sequences) from sequenced samples were compared against previously deposited organisms in the NCBI database using the Nucleotide BLAST program

(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LIN

K_LOC=blasthome) and tentatively identified based on the isolate’s closest relatives. The closest relative and its percent identity to the isolate were recorded for all sequenced isolates.

2.4.4. Assessment for Toluene Production from Isolated Bacteria

To assess whether representatives of groups in the collection of cultivated isolates were able to produce toluene, DNA from colonies that were successfully amplified with the Universal primer set, appeared to be pure cultures, and tentatively identified with a known bacterial group were transferred into anaerobic PA2D medium and analyzed for toluene production. In an

anerobic chamber (Coy Laboratory products), 9 mL anaerobic PA2D medium containing 350 mg/L phenylacetic acid was dispensed into 25 mL sterile glass serum bottles (Wheaton). The bottles were sealed with butyl rubber stoppers and aluminum crimp caps. Sterile toothpicks were used to transfer single colonies into 0.5 mL aliquots of PA2D media in microcentrifuge tubes.

Tubes were vortexed for 10 seconds. A 1 mL syringe was used to transfer the entire volume (0.5 mL) of culture in media to sterile PA2D media in serum bottles. Duplicate bottles were prepared for each strain tested. A positive control inoculated with a toluene-producing SR culture and an abiotic negative control were included. Bottles incubated at 25°C, and the presence of toluene was analyzed after 3 weeks of incubation. Turbidity was also visually observed. The strains chosen for analysis and their tentative identification (indicated by the closest related bacteria from Blastn search of a partial 16S rRNA sequence) are presented in Table 2.10. below. Stains chosen were all isolated from the previous experiments described in this chapter.

Table 2.10. Strains Inoculated into Liquid PA2D to Assess for Toluene Production

Strain ID Isolation Blastn Closest Match Percent Mediuma Identity (%) 2- 33c-1 MM 7 Micrococcus yunnanensis strain B18 100 2-19c MM 7 Micrococcus yunnanensis strain B18 100 SR1-9a SR6.5 Uncultured Azospira sp. clone CFC11 100 SR1-45a SR6.5 Uncultured Azospira sp. clone CFC11 100 8-3c VL55 5.5 Uncultured Azospira sp. clone CFC11 99.38 12-36ci-1 VL55 6.5 Uncultured Azospira sp. clone CFC11 100 2-2c MM 7 Propionicimonas sp. Y1A-10-1 97.48 12-6c VL55 6.5 Propionicimonas sp. Y1A-10-1 97.28 4-5c VL55* 7 Propionicimonas sp. Y1A-10-1 97.64 12-36cii VL55 6.5 Propionicimonas sp. Y1A-10-1 99.45 4-8c VL55* 7 Uncultured Aquabacterium sp. clone CS1 15 99.56 a See Table 2.4. for composition

2.4.5. Analytical Methods

Aqueous-phase toluene concentrations were determined using purge and trap gas chromatography as described by (Moe et al., 2018). Briefly, an Agilent Model 7820A Gas

Chromatograph (GC) equipped with a DB-624 capillary column (60 m × 0.32 mm × 1.80 μm) and a flame ionization detector (FID) was used with a temperature program that included a five minute hold at 40°C, a 20°C/minute ramp to 260°C, and a 3-minute hold at 260°C. Aqueous- phase samples were introduced to the GC utilizing a Teledyne Tekmar AQUAtek 100 auto sampler in conjunction with a Teledyne Tekmar Purge and Trap (Moe et al., 2018). pH was measured using an Orion Model 290 pH meter.

2.5. Results and Discussion

2.5.1. Cultivation and Identification of Isolates using Universal Bacterial Primers

Incubated spread plates had visible, well-isolated colonies at the time of the first transfer on to new media. The dilution plate that yielded between 15 to 43 colonies from each media variant was selected, and colonies were transferred thereafter. Table 2.11. summarizes the incubation time and dilution level from which colonies were transferred. Dilution levels representing 10-5 and 10-6 of the parent inoculum were selected for the majority of media variants. In one variant (VL55* 5.5), colonies were abundant on plates that had 10-8 of the parent inoculum.

Table 2.11. Dilution Level of Colonies Isolated after Various Days of Incubation Days of Dilution Level Colonies Media Name incubation were Isolated From MM 5.5 53 10-5 MM 7 55 10-5 VL55* 5.5 58 10-8 VL55* 7 58 10-5 VL55 5.5 62 10-7 VL55 6 90 10-5 VL55 6.5 42 10-6 VL55 5.5 GX 69 10-5 VL55 5.5 AX 59 10-6 VL55 7.2 AX 77 10-6 OVL55 5.5 78 10-6 VL55 6.5 GX 70 10-6 VL55 7 74 10-6 OVL55 6.5 55 10-7 SR6.5 21 10-4

A total of 353 colonies were grown and transferred sequentially on solid media; however, only 278 (78.75%) of these sequenced colonies appeared to be pure cultures (e.g. showing minimal overlapping peaks on the sequence electropherogram) and were tentatively identified.

Tables 2.12. and 2.13. present eight groups of organisms that the 278 isolates most closely identified with. A representative assembly or partial 16S rRNA sequence (given the quality of both forward and reverse sequences) was compared against the NCBI database using Blastn

(Table 2.12.), and the closest related organisms (including uncultured clones) along with the percent identity to the isolated strains were recorded for each group. The EZBioCloud 16S rRNA gene database was used to identify the closest related validly published organism to the isolated strains (shown in Table 2.13.). Isolates related closely (>95.98) to validly published species from the genera Bacillus, Micrococcus, Anoxybacillus, Cellulosimicrobium, Bradyrhizobium,

Azospira, and Propionicimonas. Figure 2.1. presents the relative abundances of the grouped organisms. The majority of isolates identified most closely with the genera Propionicimonas

(196 of 275 isolates) and Azospira (69 of 275 isolates) which represented 71.27% and 25.09% of the total, respectively.

Strains in all eight groups were isolated on various VL55 media variants. The strain identifying with group 1 was isolated on media solidified with washed agar and supplemented with glucose at pH 5.5. One strain from group 2 was isolated on VL55 medium with glucose and solidified with Gelzan at pH 6.5. The remaining strains were isolated on medium with glucose and solidified with washed agar at pH 7. Strains from group 3 were isolated on one media variant. The medium contained xylan and was solidified with washed agar at pH 5.5. Only one strain was isolated for group 4 and group 5. The strain from group 4 was isolated on medium at pH 6.5 containing glucose and solidified with Gelzan. The single strain in group 5 was isolated on medium at pH 5.5 containing glucose and solidified with washed agar.

The strains closely identifying with the genus Propionicimonas were divided into two subgroups. Strains within group 7 (Table 2.12.) identified most closely (99.4%) with

Propionicimonas ferrireducens Y1A-10-4-9-1T, whereas those within group 8 identified most closely (99.09%) with Propionicimonas paludicola DSM 15597T. Isolated strains in group 7 were more abundant comprising 50.91% of the total cultured strains. The strains in group 7 grew on media solidified with Gelzan and supplemented with glucose at pH 6, 6.5, and 7 and media solidified with Gelzan and supplemented with xylan at pH 5.5 and 6.5. They were also isolated on media solidified with washed agar and supplemented with glucose or xylan at pH 5.5, 6.5, 7, and 7.2. Strains in group 8 were isolated on media solidified with washed agar and supplemented with glucose at pH 5.5 and 7 and media solidified with Gelzan and supplemented with xylan at pH 5.5 and 6.5. Additional strains were isolated on media solidified with Gelzan and

supplemented with glucose at pH 5.5, 6, 6.5, and 7 and media solidified with washed agar and supplemented with xylan at pH 7.2.

All isolates related to the genus Azospira (group 6) appeared to be identical in partial 16S rRNA gene sequences. Representative sequences were selected and compared against the

EZBioCloud 16S rRNA gene database. The sequences were between 95.98-98.06% similar to

Azospira oryzae strain 6a3T and between 94.35-96.85% similar to Azospira restricta strain

SUA2T. Strains in this group were isolated on media solidified with Gelzan and supplemented with glucose at pH 6, 6.5, and 7 and media solidified with Gelzan and supplemented with xylan at pH 5.5 and 6.5. Additional strains were also isolated on media solidified with washed agar and supplemented with xylan at pH 5.5 and 7.2. This genus is further discussed in Chapter 3.

Table 2.12. Closest Representatives to Grouped Isolates Based on the Blastn Algorithm Using the NCBI Database

Number of Isolates Group in Blastn Closest Representative (top 3 Percent Number Group hits) Accession # Identity Bacillus sp. N059b KC252732.1 99.89 1 1 Bacillus sp. 3C3 GU262995.1 98.81 Bacillus kyonggiensis strain 5I MK104474.1 98.96 Micrococcus luteus strain KCOM 1845 (=ChDC B554) MT318537.1 100 2 5 Micrococcus yunnanensis strain B18 MT256062.1 100 Micrococcus aloeverae LR135583.1 100

Anoxybacillus rupiensis strain 4Cx MT350133.1 100 3 2 Anoxybacillus rupiensis strain X-015 MK418870.1 100 Anoxybacillus rupiensis strain X-08 MK418869.1 100 Cellulosimicrobium sp. strain S22 MN859997.1 100 4 1 Cellulosimicrobium sp. strain 97-5 MN592828.1 100 Cellulosimicrobium sp. strain OLMR01- 11 MN536509.1 100 Bradyrhizobium elkanii strain NAC60 MK872345.1 99.89 5 1 Bradyrhizobium elkanii strain NAC39 MK872328.1 99.89 Bradyrhizobium elkanii strain USDA 76 MN338958.1 99.89 Uncultured Azospira sp. clone MFC- B162-D07 FJ393097.1 100 6 69 Uncultured Azospira sp. clone CFC11 JF736645.1 98.86 Uncultured bacterium clone HFMBR 2- 12 JX628616.1 99.79 Propionicimonas sp. Y1A-10-1 MG775313.1 99.33 Uncultured bacterium SJA-181 AJ009505.1 99.25 7 140 Propionicimonas sp. ICHIOC20 LC132816.1 98.58 Propionicimonas paludicola strain Wd NR104769.1 98.73 Propionicimonas sp. ICHIOC20 LC132816.1 98.91 8 56 Propionicimonas paludicola strain Wd NR104769.1 98.91 Propionicimonas sp. Y1A-10-1 MG775313.1 98.82

Table 2.13. Closest Validly Published Bacterial Species to Grouped Isolates as Determined from EZ-Taxon

Number of Isolates Group in EZBioCloud Closest Validly Named Percent Number Group Organism (top hits) Accession # Identity Bacillus siralis 171544(T) AF071856 98.66 1 1 Cytobacillus oceanisediminis H2(T) GQ292772 98.59 Cytobacillus depressus BZ1(T) KP259553 98.22 Micrococcus luteus NCTC 2665(T) CP001628 99.86-100 Micrococcus endophyticus YIM 56238(T) EU005372 99.3-99.46 2 5 Micrococcus lylae NBRC 15355(T) BCSN01000086 98.5-98.61 Micrococcus antarcticus T2(T) AJ005932 98.79-99.3 Anoxybacillus rupiensis R270(T) AJ879076 99.82-100 3 2 Anoxybacillus geothermalis GSsed3(T) KJ722458 99.57-99.64 Anoxybacillus tepidamans GS5-97(T) AY563003 97.28-97.54 Cellulosimicrobium cellulans LMG 16121(T) CAOI1000358 100 Cellulosimicrobium funkei ATCC BAA- 4 1 886(T) AY501364 99.78 Cellulosimicrobium marinum RS-7-4(T) LC042213 99.12 Bradyrhizobium elkanii USDA 76(T) KB900701 100 5 1 Bradyrhizobium jicamae PAC68(T) LLXZ01000092 100 Bradyrhizobium pachyrhizi PAC 48(T) LFIQ01000091 100 Azospira oryzae 6a3(T) AF011347 96.94 6 69 Azospira restricta SUA2(T) DQ974114 95.10 Propionivibrio dicarboxylicus DSM 5885(T) FNCY01000039 94.25 Propionicimonas ferrireducens Y1A-10-4-9- 1(T) MG775313 99.4 140 7 Propionicimonas paludicola DSM 15597(T) PDJC01000001 98.81 Micropruina glycogenica Lg2(T) LT985188 96.86

Propionicimonas paludicola DSM 15597(T) PDJC01000001 99.09 56 Propionicimonas ferrireducens Y1A-10-4-9- 8 1(T) MG775313 99 Propionicicella superfundia DSM 22317(T) KE384022 97.54

Relative Abundances of Cultured Bacteria by Group

0.36 1.82 0.72 0.36 0.36

20.36 25.09

50.91

1 2 3 4 5 6 7 8

Figure 2.1. Percent Abundance of Organisms from all Cultured Bacteria by Group (see Table 2.12. and 2.13. for group affiliations).

Strains were sorted into eight groups are bacteria that identify most closely with the genera Bacillus, Micrococcus, Anoxybacillus, Cellulosimicrobium, Bradyrhizobium,

Propionicimonas, and Azospira. These genera contain species that possess a wide variety of phenotypic characteristics. The genus Bacillus, for example, contains Gram-positive bacteria.

Some species within the genus Bacillus have been found to cause human infection and illness

(e.g. anthrax). Although some species within this genus are related to human infection, others exhibit a range of physiological abilities and environmental habitats, and they have been found to live in every natural habitat, including soils and freshwater (Turnbull, 1996).The genus

Micrococcus contains bacteria that have been isolated from human skin (Kloos et al., 1974). In the research described in this thesis, there were five isolates that had partial 16S rRNA gene sequences that identified most closely (>99.86%) with the type strain of the species Micrococcus luteus (Table 2.13.). These bacteria were isolated from two different media variants (MM 7 and

VL55 6.5, further described in Table 2.4.). It is uncertain if these strains were inhabitants in the

sugar-free toluene-producing enrichment cultures because no strain has been found to inhabit soil or groundwater environments.

Representatives from the species Anoxybacillus rupiensis, which was the closest previously published relative of the isolates in group 3 based on partial 16S rRNA gene sequencing, are thermophilic and have been isolated from three hot springs in Bulgaria

(Derekova et al., 2007). The two strains isolated from the toluene-producing enrichment culture in the present study that were most closely identified with this species were isolated on low nutrient VL55 medium supplied with xylan at pH 5.5 at an incubation temperature of 25°C.

Although strains of Anoxybacillus rupiensis were reported to grow in the pH range of 5.5 to 8.5

(6.0-6.5 optimum) and degrade xylan, the species was described as obligately thermophilic with a temperature range of 35 to 67°C (55°C optimum) (Derekova et al., 2007). The isolation of these strains in this study could expand the temperature range for growth of representatives within the species. Bacteria in the genus Cellulosimicrobium are often associated with the human salivary microbiome (Wang et al., 2016). However, other species from this genus, such as

Cellulosimicrobium terreum have been isolated from soil samples (Yoon et al., 2007). The environmental range for growth appears to be variable between strains.

The genus Bradyrhizobium, relating closely to the representative strain in group 5, contains Gram-negative soil bacteria (Ormeño-Orrillo and Martínez-Romero, 2019). Similarly, strains from the genus Azospira were isolated from environmental samples such as surface- sterilized roots of Kallar grass and rice (Oryza) (Reinhold-Hurek and Hurek, 2000) and groundwater from an upgradient uncontaminated groundwater well at a Superfund Site near

Baton Rouge, Louisiana (Bae et al., 2007). The final genus that was closely identified to strains

in groups 7 and 8 was Propionicimonas. This genus contains Gram-positive bacteria.

Specifically, Propionicimonas paludicola strain WdT was isolated from anoxic plant residue from an irrigated rice-field soil in Japan (Akasaka et al., 2003). All strains except those from group 2 appear to be indigenous to soil and groundwater at the Superfund Site when compared to the isolation method of their closest relatives. Group 2, being closely related to Micrococcus spp., may not have been isolated directly from the sugar-free toluene-producing enrichment cultures because the related organisms are known to inhabit human skin and the genus did not appear to be present in previous 16S rRNA gene libraries (unpublished data).

Based on sequencing of partial 16S rRNA genes using universal bacterial primers, no isolates identified with the phylum Acidobacteria. Additionally, none of the isolates grown in liquid PA2D medium produced toluene after 3 weeks of incubation.

2.5.2. Primer Evaluation for Targeting Acidobacteria and PCR

All primers were initially tested with a positive control which consisted of DNA from a toluene-producing SR enrichment culture. Amplicons were tentatively identified by comparing a partial 16S rRNA sequence with the NCBI Blastn database. Table 2.14. summarizes the results from PCR on toluene-producing DNA (e.g. positive control) with various group- and phylum- specific Acidobacteria primers as well as the “Koribacter”-specific primer sets Tol1, Tol2, and

Tol3. Primers S1 and 31F failed to amplify DNA from the toluene-producing SR enrichment culture after multiple attempts and were not used for plate wash or colony isolate PCR. Primer set ACIDO-1492r repeatedly amplified Azospira-related strains in both positive controls and when employing template DNA from selected isolates. This non-specific amplification provided false-positive amplicons, especially since strains closely related to Azospira strains were highly abundant in the enrichment culture (unpublished data) and isolate library (25.1%, see Tables

2.12. and 2.13.). Sequences of amplicons from PCR primer sets Tol1, Tol2, and Tol3 with positive controls (SR enrichment culture community DNA) as template were >99.13% similar to an Uncultured Acidobacteria bacterium clone (closest blastn match against the NCBI database) and were closely related to Acidobacteria strain Tolsyn indicating specificity to the target group.

Table 2.14. Top Blastn Match for Positive Control DNA Amplified with Acidobacteria-specific Primer Sets

Sample ID Primer Set Used for Sequence Closest Blastn Percent PCR Purity Match Identity (%)

Positive Control ACIDO-1492r Pure Uncultured Azospira 100 sp. clone CFC11 (JF736645.1)

Positive Control Primer A-1385r Mixed Uncultured 81.21 Acidobacteria bacterium clone A6YA20RM (FJ570212.1)

Positive Control Tol1 Pure Uncultured 100 Acidobacteria bacterium clone SEW 08 085 (HQ599092.1)

Positive Control Tol2 Pure Uncultured 99.26 Acidobacteria bacterium clone Acido.wet.ACET10 (GU374504.1)

Positive Control Tol3 Pure Uncultured 99.13 Acidobacteria bacterium clone Acido.wet.ACET10 (GU374504.1)

Experimentation using Acidobacteria-specific primers did not show conclusive evidence that Acidobacteria-related organisms were present on solid media tested in plate wash PCR. The

Primer A-1385r primer set yielded a PCR product for the VL6.5 (SR6.5) 10-3 spread plate from

the sixth generation sugar-free SR enrichment culture. The partial product was sequenced and appeared to have a mixed electropherogram (peaks overlapped). It appears likely that DNA originating from multiple organisms may have been amplified with these primers. Though clearly mixed, the sequence was nevertheless analyzed via Blastn, and the best match had 72% identity with Propionicimonas. It was concluded that this primer set could amplify organisms outside of the phylum Acidobacteria.

When plate wash PCR was performed on a representative plate from all media variants using the Tol2 primer set, a PCR product of the anticipated size (~300 bp, see Table 2.7.) was observed for the VL6.5 (SR6.5) 10-2 spread plate (see Table 2.4. for composition). The sequence appeared to be mixed (overlapping peaks in electropherogram) but when subjected to blastn analysis against the NCBI database was identified to be 79% similar to Candidatus Koribacter

Ellin 345, the closest previously cultured representative. When using primer sets Tol1 and Tol3 on the same plate washed DNA extract, no PCR products were observed. Remaining isolates from the same media type (transferred off of dilution plate 10-4) were processed using both Tol2 and universal primer sets and did not identify with any Acidobacteria. After these analyses, there were no remaining plates to transfer colonies onto new media.

After around approximately 317 days of incubation, all streak plates were observed for growth a final time. A second transfer streak plate from the media variant VL7.2 AX had new colonies that were not present at day 19 of incubation (when plates were transferred a third time).

DNA was extracted, and PCR was conducted using the Tol2 primer set on one colony from this plate. A faint PCR product was observed, but the sequence was mixed (overlapping peaks in electropherogram) and BLAST analysis of the poor quality sequence did not identity it as

Candidatus Koribacter (or any Acidobacteria). From the same streak plate, 38 colonies were

individually transferred onto new media and incubated in aerobic, 5% CO2, or anaerobic atmospheres for 45 days. A total of 56 colonies (multiple morphologies were observed on one streak plate) were extracted and amplified using the Tol2 and universal primer sets. No amplification was observed with the Tol2 primers. All universal primer PCR products were sequenced, but no Acidobacteria were present. About 66% of isolates identified with the genus

Agrobacterium (with >99% similarity). Remaining isolates grouped with the genera Bacillus and

Rhizobium (based on blastn against the NCBI database).

The genus Agrobacterium contains eight Gram-negative, soil-inhabiting species. They are commonly isolated near plant roots, tubers, or underground stems (Matthysse, 2006).

Additional studies proposed the inclusion of species from the genus Agrobacterium in the genus

Rhizobium due to the similarity of their 16S rRNA genes (Matthysse, 2006). Bacillus species, as described above, are widely distributed in soils and freshwater (Turnbull, 1996).

2.6. Conclusion

Although no Acidobacteria-related strains were isolated from the toluene-producing SR enrichment culture, there were 8 different groups of bacteria tentatively identified as being most closely related to seven different genera. Strains most closely related to the genera Azospira and

Propionicimonas (based on partial 16S rRNA gene sequences) were isolated most frequently on the solid media reported here and were also abundant in the original sugar-free toluene- producing inoculum (unpublished data). In initial tests in PA2D media, representatives from the various groups of isolates did not produce toluene during a 16-day incubation period. Further research is needed to clarify what role these bacterial may play (directly or indirectly) in toluene production, toluene consumption, or toluene tolerance.

CHAPTER 3. CHARACTERIZATION OF NOVEL AZOSPIRA ISOLATES

3.1. Background of the genus Azospira

The genus Azospira was first described by Reinhold-Hurek and Hurek (2000) to distinguish a lineage of phylogenetically and phenotypically unique, nitrogen-fixing bacteria from other strains originally belonging to the genus Azoarcus sensu lato within the

Betaproteobacteria (Reinhold-Hurek et al., 1993). Currently, the genus Azospira contains two validly published species, Azospira oryzae (Reinhold-Hurek and Hurek, 2000) (type strain=

6a3T, =DSM 21223, =CCUG 45016, =LMG 9096) and Azospira restricta (Bae et al., 2007) (type strain= SUA2T, =NRRL B-41660 =DSM 18626, =LMG 23819).

Azospira oryzae 6a3T was isolated from surface-sterilized roots of Kallar grass, and additional strains have been isolated from rice (Oryza). Phylogenetic analyses of almost complete 16S rRNA sequences confirmed that the taxon initially described as Azoarcus sensu lato was not monophyletic, and bacterial isolates previously classified under this taxonomic ranking were divided among three new genera, one being Azospira which at the time of the initial genus description contained the single species Azospira oryzae (Reinhold-Hurek and

Hurek, 2000). In studies exploring perchlorate-reducing microorganisms in both pristine and contaminated soils and sediments, a bacterium initially described as Dechlorosoma suillum strain

PS was isolated from a primary treatment lagoon of swine waste (Achenbach et al., 2001). Tan and Reinhold-Hurek (2003) compared Dechlorosoma suillum strain PS and Azospira oryzae, strains which possess 99.9% 16S rRNA gene sequence identity, in a polyphasic taxonomic approach. Results were identical except for the use of perchlorate as a terminal electron acceptor by Dechlorosoma suillum strain PS. It was concluded that the bacteria belonged to the same

species with Dechlorosoma suillum considered a later subjunctive synonym of Azospira oryzae

(Tan and Reinhold-Hurek, 2003).

Bae et al. (2007) isolated a novel, nitrogen-fixing bacterium designated as SUA2T during a study aimed at characterizing the microbial population in groundwater from an upgradient uncontaminated groundwater well at the PetroProcessors of Louisiana, Inc. Superfund Site located near Baton Rouge, Louisiana. The bacterium was isolated on R2A agar that was incubated aerobically at room temperature. This novel strain is distinct from Azospira oryzae strains 6a3T and PS in substrate utilization, cellular fatty acid composition, and mol% G+C, leading to the description of Azospira restricta as a novel species and emendment of the genus

Azospira (Bae et al., 2007).

At a higher taxonomic level, the genus Azospira along with the related genera

Rhodocyclus (type genus), Propionibacter, and Propionivibrio falls within the recently emended family Rhodocyclaceae within the order Rhodocyclales of the class Betaproteobacteria (Boden et al., 2017). The order Rhodocyclales contains several genera able biodegrade toluene under anaerobic conditions. For example, members of the genera Azoarcus (Anders et al., 1995; Zhou et al., 1995), Dechloromonas (Chakraborty et al., 2005; Achenbach et al., 2001; Horn et al.,

2005; Wolterink et al., 2005), Georgfuchsia (Weelink et al., 2009) and Thauera (Anders et al.,

1995) were reported as anaerobic toluene degraders. Members of the newly described genus

Aromatoleum are able to biodegrade toluene under anaerobic and aerobic conditions (Rabus et al., 2019). The order also contains the genus Rugosibacter which reportedly grows well on benzene and weakly on toluene under aerobic conditions (Corteselli et al., 2017).

As described in chapter 2 of this thesis, several (69) strains were isolated that were most closely related to Azospira strains on the basis of partial 16S rRNA gene sequences. Because the

partial 16S rRNA gene sequences of the new isolates were identical to partial 16S rRNA gene sequences that had high representation in the toluene-producing sugar-free SR enrichment cultures (unpublished data), experiments described in this chapter were conducted to clarify the strains’ taxonomic position and explore what role they may play in toluene production or consumption.

3.2. Materials and Methods

3.2.1. Bacterial Strains and Preservation

Three bacterial strains isolated from the toluene-producing, sugar-free, SR enrichment culture were selected for more detailed characterization. The strains were arbitrarily designated as Az-1, Az-2, and Az-3. Strains Az-1 and Az-2 were isolated from the second generation of sugar-free culture but on different media. Az-1 was isolated aerobically on VL55 media adjusted to pH 7 (VL7), solidified with Gelzan, and supplemented 0.36 g/L glucose. Strain Az-2 was isolated aerobically on VL55 media adjusted to pH 5.5 with xylan and solidified with washed agar. Strain Az-3 was isolated from the sixth generation of the sugar-free SR enrichment culture aerobically on VL55 media adjusted to pH 6.5 (SR6.5) with glucose and solidified with Gelzan.

Strains Az-1 and Az-2 originated from a 10-6 dilution spread plate and Az-3 originated from a 10-

5 dilution spread plate. Following isolation, all three strains were routinely grown on R2A agar

(Difco) under aerobic conditions at 25°C or 37°C.

For preservation, single colonies grown on R2A were inoculated into 50 mL R2B (see section 3.3.1), incubated at 25°C for 4 days (at which time turbid growth was observed), mixed with 15% (v/v) filter-sterilized glycerol, and then 1 mL aliquots were dispensed into sterile cryovials which were stored at -80°C.

For comparative purposes, Azospira oryzae 6a3T (=DSM 21223), Azospira oryzae PS

(formerly Dechlorosoma suillum PS, =DSM 13638), and Azospira restricta SUA2T (= DSM

18626) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen

GmbH (DSMZ, Braunschweig, Germany). The reference strains, obtained as lyophilized cultures were resuspended with 0.5 mL liquid R2B, and incubated for 30 minutes at room temperature, mixed, and then streaked on to R2A. Following growth, single colonies were transferred to R2B, grown, and preserves were prepared as described above for strains Az-1, Az-2, and Az-3.

3.2.2. 16S rRNA Gene Sequencing

PCR products of all strains amplified using universal bacterial primers 27f YM (Frank,

2008) and 1492r (Lane, 1991) were sequenced via Sanger sequencing at Eton Bioscience, Inc.

(as described in section 2.4.2) using primers 27f YM, 1492r, 1114f (Rainey, 1996), and 519r

(Rainey, 1996) to obtain almost complete 16S rRNA gene sequences. The BioEdit v.7.2.5

Sequence Alignment Editor (Hall, 1999) was used to manually assemble and align sequences.

Primers 27f YM and 1492r (used during sequencing) were trimmed from the consensus sequences. Pairwise sequence similarities between strains Az-1, Az-2, and Az-3 and various type strains were calculated using the facilities of the EZBioCloud database (Yoon et al., 2017).

3.3. Media Preparation 3.3.1. R2A and R2B

All strains were grown on R2A agar (Difco), prior to making preserves. R2B, or “R2- broth”, was prepared by combining the constituents found in R2A agar but excluding agar. This included the following constituents (per liter): yeast extract, 0.5 g; proteose peptone No. 3, 0.5 g; casamino acids, 0.5 g; dextrose, 0.5 g; soluble starch, 0.5 g; sodium pyruvate, 0.3 g; dipotassium

phosphate, 0.3 g; MgSO4, 0.05 g. It was brought to a boil and autoclaved. Once cooled, the media was dispensed into sterile serum bottles.

3.3.2. VM Ethanol (VME)

VM ethanol medium, as described by Reinhold-Hurek and Hurek (2000), was prepared by combining the following constituents (per liter): K2HPO4, 0.6 g; KH2PO4, 0.4 g; NH4Cl, 0.5 g; MgSO4·H2O, 0.2 g; NaCl, 1.1 g; CaCl·2H2O, 0.026 g; MnSO4·H2O, 0.01 g; Na2MoO4·2H2O,

0.002 g; Fe(III)-EDTA, 0.066 g; yeast extract, 1 g; Bacto-tryptone, 3 g. The medium was autoclaved for sterilization. The final pH was 6.8 after autoclaving. Once completely cooled,

0.6%, v/v filter-sterilized ethanol was added to the medium prior to dispensing. Bacto Agar was added at a concentration of 15 g/L when used as a solid growth medium.

3.4. Comparative Testing

Cryopreserved cultures were thawed and inoculated (0.2 %, v/v) into sterile liquid R2B and incubated at 37°C for a period of 2 to 3 days prior to use in comparative testing. The incubation period was sufficient to achieve turbid cultures. New cryopreserves were used for each test. All tests were performed in duplicate, unless otherwise stated, and abiotic negative controls were included. Unless stated otherwise, all six strains (three new isolates plus three reference strains) were used in all tests.

3.4.1. Colony Morphology

Colony morphology of the six strains was observed following growth on VM ethanol medium (solidified with 15 g/L agar) after incubated at 37°C for 4 days and on R2A incubated at

30°C for 7 days. The color, shape, margin, and elevation were recorded according to classifications in Smibert and Krieg (1981). The diameters of well isolated colonies were

measured with imaging performed using a Celestron Digital Microscope and MicroCapture Pro software.

3.4.2. Cell Morphology

Cellular characteristics were observed using scanning electron microscopy in the

Louisiana State University Shared Instrumentation Facility. A 0.4 µm sterile tech polycarbonate membrane filter (13mm) was placed inside a syringe fitted with a rubber neck and washer. The fixing solution was prepared by combining 4% OsO4, 27%, v/v; 16% formaldehyde, 13.4%, v/v;

25% glutaraldehyde, 8.6%, v/v; and deionized water, 51.3%, v/v. Samples were combined with an equal volume of fixing solution in a plastic weigh dish. With the membrane filter detached from the syringe, the entire volume of sample and fixing solution was collected in the syringe.

Reattaching the membrane, the solution was pushed through the membrane to fix the cells on the membrane’s surface. The samples sat at room temperature for 20 minutes prior to washing with deionized water three times.

Once the fixing solution was removed, cells were dehydrated by rinsing with 50%, 70%, and then 90% ethanol (15 minutes each). Using 100% ethanol, the cells were washed three times and allowed to sit for 15 minutes in between each wash. The membrane filters were removed from the syringe and submerged in a 1:1 solution (1 mL total volume) of 100% ethanol and hexamethyldisilazane (HMDS) and allowed to incubate for 15 minutes. The solution was removed and 1 mL 70% HMDS (with 30% ethanol) was added to the membrane. After 15 minutes, the solution was removed and 1 mL 100% HMDS was added to the membranes. The membranes incubated for 15 minutes, the liquid was removed, and were then soaked in 100%

HMDS a second time. The entire volume of liquid was removed from the dishes, and the membranes were allowed to dry overnight.

Cells were sputter coated using an EMS 550X Sputter Coater (Electron Microscopy

Sciences). Samples were then loaded into a JSM-6610/6610V scanning electron microscope for imaging. Cell length, width, and shape were recorded.

3.4.3. Gram Staining

Gram staining was performed using a BD Gram Stain Kit following the manufacturer’s recommended protocol in conjunction with heat-fixed smears prepared after strains were grown at 37°C for 3 days.

3.4.4. Presence of Catalase

Test for the presence of the catalase enzyme was performed following the procedure described by Smibert and Krieg (1981) following growth on R2A, incubated at 37°C for 4 days.

A sterile inoculating loop was used to pick up a large amount of biomass from the plate and spread onto a clean glass slide. After, 3% hydrogen peroxide was added to cover the biomass.

The slides were observed for bubbling immediately after adding hydrogen peroxide and after 5 minutes. Samples exhibiting gas bubble formation were scored as catalase positive.

3.4.5. Presence of Oxidase

Test for the presence of the oxidase enzyme was performed according to Tarrand and

Grӧschel (1982) after growth on R2A incubated at 37°C for 3 days. The oxidase test was prepared by placing a Whatman (Cat No 1001-042) 42.6 mm, circular filter paper inside a sterile plastic petri dish (VWR). A solution of 1% w/v tetramethyl-p-phenylenediamine in dimethylsulfoxide (DMSO) (1% TMP-DMSO) was prepared under the fume hood. Using a sterile pipette, 0.5 mL of the 1% TMP-DMSO was transferred to the filter paper. A sterile cotton-tipped applicator (Fisher) was used to collect a portion of biomass from the plate. The cotton tip was then tamped lightly on the wet filter paper. The reaction was observed after 10, 15

and 30 seconds on the cotton tip. An oxidase positive sample would show a color change to purple-blue whereas an oxidase negative sample would show no color change.

3.4.6. Salt Tolerance

Salinity range for growth was assessed in VM ethanol broth. After incubating for 2 days at 25°C in liquid R2B, 0.2%, v/v of the cultures were transferred into sterile liquid VM ethanol media. Liquid VM ethanol contained either 1% or 2%, w/v filter sterilized NaCl. The bottles incubated at 37°C. Growth at elevated NaCl was visually determined by an increase in turbidity after 3 and 6 days of incubation. All bottles were compared to an abiotic negative control.

3.4.7. Temperature Range

The temperature range for growth was assessed in VM ethanol broth. Strains were inoculated, at a concentration of 0.2%, v/v, into VME bottles and incubated at either 15°C, 20°C,

23°C, 30°C, 37°C, and 40°C for a total of 11 days. The absorbance at 600 nm was measured for each strain in duplicate using an Evolution 60 spectrophotometer (Thermo Scientific) after 3 days and 11 days. The absorbances at each temperature were plotted to determine the optimal temperature for growth.

3.4.8. pH Range

The pH range for growth was assessed in liquid VL55 media with 0.5 g/L succinic acid

(added as sodium succinate) adjusted to pH 5.5, 6, 7, 8, 9, and 10 using 200 mM NaOH/100 mM

KOH prior to sterilization. Sterile media was dispensed into sterile borosilicate glass tubes with ventilated caps to allow oxygen transfer. Strains were inoculated at a concentration of 0.17%, v/v into the tubes and incubated at 25°C for up to 9 days. Absorbance as an indication of growth was measured at 600 nm after 3, 7, and 9 days of incubation.

3.4.9. Biological and Physiological Properties using API 20 NE Test Strips

To assess for the presence of various enzymes and substrate utilization, strains were analyzed on API 20 NE test strips (bioMérieux) following the manufacturer’s protocol summarized below. Strains were initially grown on R2A for 10 days at 25°C. Colonies were transferred into 2 mL of 0.85%, w/v sterile saline using a sterile 1 µL inoculating loop to achieve a turbidity equivalent to 0.5 McFarland. The McFarland standard was prepared by combing 0.05 mL 1% BaCl2 with 9.95 mL 1% H2SO4 in a sterile tube. The accuracy of the standard was verified by measuring the absorbance at a wavelength of 625 nm. A 0.5 McFarland Standard has an absorbance of 0.08-0.1 at this wavelength (DALYNN Biologicals, 2014).

The saline suspension was distributed into tests NO3 to PNPG using a sterile pipette.

Approximately 200 µL of the remaining suspension was added to the ampule of API AUX

Medium and homogenized. The tubes and cupules of tests GLU to PAC were filled with the suspension. Mineral oil was then added to the cupules GLU, ADH, and URE to form a convex meniscus. The inoculated strips incubated at 30°C for 24 hours.

The strip was scored according to the Reading Table in the API 20 NE Instruction

Manual (REF 20 050). TRP test was performed by adding one drop of JAMES reagent. The tube was observed for an immediate color change. The assimilation tests were observed for the presence of an opaque cupule, which indicates turbid growth. Once the readings were made, the strips were incubated a second time at 30°C for 24 hours, and all tests except for TRP and GLU were read again. Assessment for the reduction of nitrates to nitrites/nitrogen, esculin hydrolysis, and malic acid assimilation were not performed using API 20 NE test strips and were analyzed using different methods.

3.4.10. Test for Esculin Hydrolysis

The assessment for esculin hydrolysis was performed according to Hussain Qadri et al

(1980). The test media was prepared by combining the following constituents (per liter): esculin,

5 g; ferric ammonium citrate, 0.5 g; sodium chloride, 8 g; K2HPO4, 0.4 g; and KH2PO4, 0.1 g with deionized water. The media was adjusted to a pH of 5.6 with 200 mM NaOH/100 mM

KOH, sterilized, and dispensed into 0.2 mL quantities in sterile 0.5 mL microcentrifuge tubes.

All strains were washed with 0.9%, w/v NaCl, centrifuged at 15,000 × g in 1.5 mL centrifuge tubes, and the cell pellet was resuspended in test media to achieve a density equivalent to no. 3

McFarland. A No. 3 McFarland standard was prepared by combining 0.3 mL 1% BaCl2 with 9.7 mL 1% H2SO4 (DALYNN Biologicals, 2014). Samples incubated in a 35°C water bath and were visually observed for a color change to dark brown or black at 15, 30, 60, and 120 minutes. A positive test for esculin hydrolysis resulted in a color change to dark brown or black when compared against an abiotic negative control.

3.4.11. Substrate Utilization and Nutritional Profile

Because the novel strains were isolated on VL55 variants, the ability for cells to utilize additional substrates was determined in aerobic VL55 media adjusted to pH 7 (VL7). Media supplemented with various carbon sources was dispensed into sterile borosilicate glass tubes with ventilated caps to allow oxygen supply. Strains were grown for 5 days in liquid R2B medium at 37°C prior to inoculation at 0.17% (v/v). Substrates included (500 mg/L) acetic acid,

L-aspartic acid, L-glutamic acid, β-hydroxybutyric acid, α-ketoglutaric acid, D,L-lactic acid, L- malic acid, L-proline, propionic acid, putrescine, and succinic acid (generally added as sodium salts). Inoculated tubes containing VL7 media without carbon substrates served as negative controls. Growth was determined by measuring absorbance at 600 nm after 4, 7, and 18 days

incubation at 25°C. Tubes were scored positive if A600 was ≥0.05 compared to inoculated controls with no substrate added.

3.4.12. Production of PHB (poly-β-hydroxybutryate)

The presence of poly-β-hydroxybutyrate (PHB) was determined following the protocol described by Smibert and Krieg (1981). Strains were grown in R2B medium for 3 days at 30°C prior to biomass collection. A total of 50 mL (for reference strains) and 150 mL (new isolates) was centrifuged at 3000 × g at 4°C for 30 minutes in conical glass centrifuge tubes. The supernatant was decanted to leave 1 mL of liquid and the cell pellet, and 9 mL of 5% sodium hypochlorite was added to the tube to digest the cell components. The solution was homogenized and incubated for 24 hours at room temperature. The mixture was centrifuged to collect the PHB granules, and the supernatant was discarded. The granules were washed by suspending in distilled water, centrifuged, and then discarding the supernatant. This process was repeated once more with water then twice with acetone and twice with diethyl ether. Tubes were briefly (10 min) dried in an oven to evaporate the remaining liquid. Once dried, 2 mL concentrated sulfuric acid was added, and the tubes were placed in a boiling water bath for 10 minutes to convert PHB to crotonic acid. Samples were placed in a quartz cuvette, and the absorbance spectrum was determined at 5-nm increments from 215 to 255 nm against a blank of plain concentrated sulfuric acid. A positive test, the presence of crotonic acid, was indicated by an absorption peak at 235 nm.

3.4.13. Denitrification/Nitrate reduction

The ability of strains to use nitrate as an electron acceptor was assessed in anoxic VL7 medium supplemented with 0.5 g/L β-hydroxybutyric acid instead of glucose and 0.595 g/L

- NaNO3 (98 mg/L NO3 -N). The medium was prepared anoxically by boiling for 5 minutes and

then purging the headspace with 95% N2: 5% CO2 for 5 minutes prior to autoclaving. It was dispensed into sterile serum bottles in an anerobic chamber. Once removed, the gas headspace of each serum bottle was purged for 2 minutes with 95% N2: 5% CO2. Bottles were inoculated 2%, v/v in the anaerobic chamber and incubated at 25°C. Nitrate and nitrite were measured using an

ICS-2000 ion chromatograph (Dionex) equipped with an IonPac AS11-HC column and ASRS-

ULTRA II (4 mm) suppressor and conductivity detector after 49 days. The sample injection volume was 100 µL. Elution was performed with KOH (1.5 mM for 10 minutes then ramping to

30 mM at a rate of 1.6 mM/min) at a flow rate of 1.5 mL/min at 30°C. Retention times and peak

- - areas were compared with those of standards, which included NO2 -N and NO3 -N concentrations of 1, 5, 10, 50, and 100 mg/L.

3.4.14. Extraction and Preparation of Genomic DNA

To prepare high molecular weight DNA suitable for sequencing via the PacBio Sequel II system, DNA extraction was performed using a GenElute™ Bacterial Genomic DNA Kit following the manufacturers protocol as described below. After turbidity was verified in R2B, 30 mL (for reference strains) and 50 mL (for new isolates) of culture was pelleted by centrifugation at 3000 × g at 4°C for 30 minutes. Supernatant was decanted, and the cell pellet was resuspended in 180 µL of Lysis Solution T and 20 µL of Proteinase K (20 mg/mL). The solution was mixed and incubated at 55°C to initiate cell lysis. After 30 minutes of incubation, 200 µL of Lysis

Solution C was added to the sample tube and mixed by inverting several times. The solution was then placed back in a 55°C water bath for 10 minutes. During incubation, the binding column was prepared by adding 500 µL of Column Prep Solution to the pre-assembled GenElute

Miniprep Binding Column and centrifuged at 12,000 × g for 1 min. The eluate was discarded.

After incubation, 200 µL of molecular grade ethanol was added to the lysate and inverted to mix.

The entire contents of the sample tube were transferred into the prepared GenElute Miniprep

Binding Column and centrifuged at 6,500 × g for 1 minute. The collection tube containing the eluate was discarded and the binding column was placed in a new, clean 2 mL tube. The DNA was washed by adding 500 µL Wash Solution 1 to the column and centrifuging at 6,500 × g for

1 minute. It was washed a second time by adding 500 µL Wash Solution (with ethanol) to the dry column and centrifuged for 3 minutes at maximum speed (12,000-16,000 × g). The column was placed in a new sterile tube, and 100 µL TE buffer (pH 8) was added to the column. It incubated for 5 minutes at room temperature then centrifuged for 1 minute at 6,500 × g. Eluted DNA was transferred to a 0.2 mL, polypropylene (low binding) MicroAmp 8-tube strip.

DNA quality was determined by gel electrophoresis on a 1% (w/v) agarose gel. Gel markers were prepared by combining 2 µL deionized water, 0.5 µL purple gel loading dye (New

England BioLabs cat. no. B7025S), and 0.5 µL Lambda DNA-HindIII Digest (New England

BioLabs cat. no. N3012S). Samples were prepared by combining 0.5 µL deionized water, 0.5 µL purple gel loading dye, and 3 µL DNA extract. The gel ran for 45 minutes at 72 to 74 V. Gels were imaged on a BioRad gel imaging system using UV transillumination and viewed using

Quantity One 4.4.0. Samples were assessed by verifying that the DNA bands were larger than 23 kbp and did not show any smearing (indicative of DNA shearing or degradation) on the gel image.

A Nanodrop 2000 (Thermo Scientific) was utilized to assess the purity of extracted DNA.

With a sterile 10 µL pipette, 2 µL DNA extract was pipetted onto the stand of the Nanodrop

2000. TE buffer was used as a blank. The 260/280 and 260/230 readings were recorded

DNA quantity was determined using a Qubit 3.0 Fluorometer in conjunction with a Qubit dsDNA HS assay kit (Invitrogen) following the manufacturer’s recommended protocol. Working

solution was prepared by diluting dsDNA HS Reagent 1:200 in dsDNA HS Buffer in a clean plastic tube to make a final volume of 200 µL per sample. Between 190 and 199 µL working solution was dispensed into clean 0.5 mL tubes. Standards were prepared by combining 190 µL working solution with 10 µL Qubit dsDNA HS Standard 1 or 2. Samples were prepared by combining 1 to 3 µL with the working solution. Samples tubes were vortexed for 2 to 3 seconds then incubated for 2 minutes at room temperature before analysis. The concentration (in µg/µL) was recorded for all samples.

3.4.15. Cellular Fatty Acids

Analysis of cellular fatty acids was performed by the Identification Services of the

Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig,

Germany). Prior to analysis, strains were regrown on R2A for 3 days at 37°C. Cellular fatty acids were determined using the Sherlock Microbial Identification System (MIDI) with fatty acid identification using the TSA40 4.10 library.

3.4.16. Experimental Design for Toluene Consumption

The ability of strains to utilize toluene was assessed in anoxic VL7 medium supplemented with 595 mg/L NaNO3 and 100 mg/L toluene or 0.05%, v/v of 2%, v/v toluene in heptamethylnonane supplied from filter sterilized, anaerobic stocks. Media was prepared anaerobically by boiling the medium for 5 minutes and then purging the headspace with 95% N2:

5% CO2 for 5 minutes prior to autoclaving. It was dispensed into sterile glass serum bottles in an anaerobic chamber. Bottles were capped and sealed with Teflon lined butyl rubber stoppers. The gas headspace was purged in each bottle for 2 minutes with 95% N2: 5% CO2. Bottles were inoculated with 0.2% (v/v) cultures inside an anaerobic chamber. Toluene concentrations were measured via gas chromatography after 3 and 7 days of incubation at 25°C.

3.5. Results

The nearly complete 16S rRNA gene sequences determined for strains Az-1, Az-2, and

Az-3 were identical (see Appendix B for sequence in fasta format). The ten most closely related

type strains as determined using the EZ BioCloud 16D-based ID are presented in Table 3.1.

below. The two most closely related type strains to Az-1, Az-2, and Az-3 were Azospira oryzae

6a3T and Azospira restricta SUA2T with pairwise similarities of 96.94 and 95.10%, respectively.

This is below the 97% (or higher) threshold for classification as a separate species (Stackebrandt

and Goebel, 1994; Stackebrandt and Ebers, 2006; Meier-Kolthoff et al., 2013). Other species all

had pairwise similarities below 95% (Table 3.1.).

Table 3.1. Pairwise similarity between strains Az-1, Az-2, and Az-3 and the most closely related type strains in the EZBioCloud database

Species Type Strain Accession Mismatches Pairwise Similarity (%) Azospira oryzae 6a3 AF011347 44/1436 96.94 Azospira restricta SUA2 DQ974114 71/1450 95.10 Propionivibrio dicarboxylicus DSM 5885 FNCY01000039 84/1462 94.25 Oryzomicrobium terrae TPP412 KR296798 86/1462 94.12 Rhodocyclus tenuis DSM 109 D16208 87/1458 94.03 Azonexus hydrophilus DSM 23864 AUCE01000006 89/1460 93.90 Dechloromonas denitrificans ATCC BAA-841 LODL01000012 90/1460 93.84 Propionivibrio limicola GolChi1 AJ307983 91/1461 93.77 Dechloromonas hortensis MA-1 AY277621 94/1459 93.56 Azovibrio restrictus DSM 23866 AUCH01000043 99/1460 93.22

After 7 days incubation on R2A agar at 30°C, strains Az-1, Az-2, and Az-3 all formed

circular, convex, and smooth, cream to orange, translucent colonies with an entire margin

approximately 1 mm in diameter. Under the same incubation conditions, Azospira oryzae 6a3T

formed larger colonies approximately 3 mm in diameter that were translucent orange colored,

irregular, and umbonate with an entire margin. Azospira oryzae PS had irregular, umbonate,

cream to yellow translucent colonies approximately 2 mm in diameter with an entire margin.

Azospira restricta SUA2T formed circular, smooth, convex, cream to translucent colonies approximately 2 mm in diameter with an entire margin.

After 4 days at 37°C on VME medium, strains Az-1, Az-2, and Az-3 formed smooth, circular, convex colonies that were translucent pink to salmon colored with an entire margin.

Colonies were approximately 2 or 3 mm in diameter. Colonies of reference strains were around the same size or slightly smaller than Az-1, Az-2, and Az-3. Azospira oryzae strains 6a3T and PS formed circular, smooth, convex colonies that were translucent pink to salmon colored with an entire margin. Colonies from strain 6a3T were approximately 2 mm in diameter, whereas colonies from strain PS were approximately 3 mm in diameter. Azospira restricta SUA2T formed colonies that were circular, smooth, convex, translucent, and green to yellow in color. Colonies were approximately 1 mm in diameter with an entire margin.

After 3 days at 37°C on R2A, all six strains stained Gram-negative and were observed to be curved rods. From SEM, it was observed that for Az-1 and Az-3, cells lengths were approximately 2 µm, and cell widths were approximately 0.5 µm (Figure 3.1.). Az-2 was not imaged via SEM. Cells of Azospira oryzae 6a3T had lengths approximately 2 µm and widths approximately 0.4 µm. Azospira restricta SUA2T had cell lengths approximately 1.5 µm and widths 0.5 µm.

Figure 3.1. Scanning electron microscope images of Az-3 (left) and Az-1 (right) cells. Scale bar indicates 1 micron. All strains were oxidase positive after incubating for 3 days at 37°C on R2A, as indicated by color change to purple on the surface of a sterile cotton swab with TMP-DMSO. Strains Az-1,

Az-2, and Az-3 were all catalase negative after growth for 4 days at 37°C on R2A; whereas, A. oryzae strain 6a3T and PS and A. restricts SUA2T were catalase positive.

After 6 days at 37°C in liquid VME medium, all strains except A. restricta SUA2T grew well with 1% (w/v) NaCl. Strains Az-1, Az-2, Az-3, and Azospira oryzae strains 6a3T and PS showed turbid growth when compared to an abiotic negative control while SUA2T had no visible growth. Under the same growth conditions but with 2%, w/v NaCl, none of the six strains showed turbidity after 6 days incubation.

All six strains showed growth at the temperature range between 15 and 40°C in VME medium after 11 days. Strains Az-1, Az-2, and Az-3 grew optimally between 30 and 40°C after 3 days. They showed weaker growth at 15 and 22°C after 11 days. Figure 3.2. shows temperature

T versus A600 for these strains after 3 days incubation. A. oryzae trains 6a3 and PS grew at the temperature range between 15 and 40°C after 11 days and had optimal growth between 30 and

40°C after 3 days. A. restricta SUA2T showed turbid growth at the temperature range between 15 and 40°C after 11 days and had optimal growth between 22 and 40°C. Figure 3.3. shows

T T temperature versus A600 for strains 6a3 , PS, and SUA2 after 3 days of incubation. The range of growth for all strains is between at least 15°C and at least 40°C.

Temperature Range for Az-1, Az-2, Az-3 after 3 days 0.6

0.5

0.4

0.3 Az-1 3d Az-2 3d 0.2 Az-3 3d 0.1

0 Average nm at600 Absorbance Average 10 15 20 25 30 35 40 45 Temperature (°C)

Figure 3.2. Average Absorbance of Strains Az-1, Az-2, and Az-3 at different Temperatures

Temperature Range for strain 6A3T, PS, and SUA2T after 3 days 0.3

0.25 0.2

0.15 Strain 6a3 3d 0.1 Strain PS 3d Strain SUA2 3d 0.05

10 15 20 25 30 35 40 45 Average nm at600 Absorbance Average Temperature (°C)

Figure 3.3. Average Absorbance of Strains 6a3T, PS, and SUA2T at different Temperatures

After 9 days of incubation at 25°C in VL-medium supplemented with 0.5 g/L succinic acid, strains Az-1, Az-2, and Az-3 grew within the pH range of 5.5 to 9 but not at pH 10.

Optimal pH range was between pH 5.5 and 6 after 3 days A. oryzae strains 6a3T and PS grew over the pH range of 5.5 to 10 (pH 6 to 10 was optimum) under the same incubation conditions.

A. restricta SUA2T grew over the pH range of 7 to 9 after 11 days and showed optimal growth at pH 8 after 3 days.

All six strains were unable to assimilate N-acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-glucose, D-maltose, D-mannitol, D-mannose, phenylacetic acid, potassium gluconate, or trisodium citrate after 48 hours incubation on API 20NE test strips. All were negative for indole production, fermentation of D-glucose, the presence of arginine dihydrolase, protease, and β-galactosidase. Strains Az-1, Az-2, and Az-3 were all positive for urease, indicated by a color change from yellow to orange/pink/red, while both A. oryzae strains 6a3T and PS and A. restricta SUA2T were negative for the presence of urease.

As determined by the method described in Hussain Qadri et al (1980), all six strains were unable to hydrolyze esculin.

All strains were unable to utilize putrescine but were able to use succinic acid and β- hydroxybutyric acid when incubated at 25°C for 18 days in VL7 medium. Under the same incubation conditions, Az-1, Az-2, and Az-3 were unable to utilize L-aspartic acid, L-glutamic acid, D,L-lactic acid, propionic acid, and L-proline. They could utilize acetic acid, α-ketoglutaric acid, and L-malic acid, however. A. oryzae strains 6a3T and PS could utilize L-aspartic acid, acetic acid, L-glutamic acid, α- ketoglutaric acid, D,L-lactic acid, L-malic acid, and propionic acid, but were unable to utilize L-proline. A. restricta SUA2T was able to utilize aspartic acid,

D,L-lactic acid, L-proline, and propionic acid but not acetic acid, L-glutamic acid, α-ketoglutaric 60

acid, or malic acid. A table summarizing the substrate utilization profiles from this experiment is presented in Table 3.2. below.

Table 3.2. Substrate Utilization Profile for all Strains. (+) positive, (-) negative

Substrate Az-1 Az-2 Az-3 6a3T PS SUA2T Putrescine ------D,L-lactic acid - - - + + + α-ketoglutaric acid + + + + + - L-malic acid + + + + + - L-proline - - - - - + L-glutamic acid - - - + + - L-aspartic acid - - - + + + Succinic acid + + + + + + Β-hydroxybutyric acid + + + + + + Propionic acid - - - + + + Acetic acid + + + + + -

All six strains produced PHB storage granules during incubation at 30°C for 3 days indicated by the presence of crotonic acid which was confirmed by an absorbance peak at 235 nm. Plots showing absorbance across a wavelength spectrum for all samples are shown below.

Absorbance Spectrum for strains Az-1 and SUA2T 0.25

0.2

0.15

0.1 Az-1 avg

Absorbance SUA2 avg 0.05

0 210 220 230 240 250 260 Wavelength (nm)

Figure 3.4. Absorbance vs. Wavelength for Strains Az-1 and SUA2T. The dashed line denotes 235 nm, the maximum absorbance for the detection of crotonic acid.

Absorbance Spectrum for Strains Az-2, Az-3, 6a3T and PS 2.5

1.5 6a3

1 PS

Absorbance Az-2 0.5 Az-3 0 210 220 230 240 250 260 Wavelength (nm)

Figure 3.5. Absorbance vs. wavelength during the measurement of crotonic acid as an indicator of PHB production by strains 6a3T, PS, Az-2, and Az-3. The dashed line denotes 235 nm, the maximum absorbance for the detection of crotonic acid. Assessment for cells to reduce nitrate was verified by growth as assessed by measuring

A660 after 3 and 7 days of incubation as well as nitrate and nitrite quantification via ion chromatography (IC) after 49 days incubation. Nitrate and nitrite were undetected or present in

trace concentrations (<0.4 mg/L) after 49 days of incubation at 25°C in anoxic VL7 medium, indicating essentially complete nitrate reduction.

When cells were grown on R2A for 3 days at 37°C, cellular fatty acid profiles were similar for strains Az-1, Az-2, and Az-3 (Table 3.3.). For all three isolates, the cellular fatty acid present in the highest proportion was C16:0 (48.8 to 52.6%), followed by the compound identified in the Sherlocked Microbial Identification System (MIDI) as Summed Feature 3 (C16:1 ω7c and/or C15:0 iso 2OH) at abundances that ranged from 30.3 to 34.1%. and C18:1 ω7c at abundances that ranged from 5.2 to 6.2%. C17:0 cyclo was detected in all three strains but at proportions that were more variable, ranging from 1.1 to 6.3%. The following cellular fatty acids were present in lower amounts (<2% each): C8:0 3OH, C10:0 3OH, C14:0, C14:1 ω5c, C15:1 ω6c,

C17:0, C18:1 ω9c, C15:0, and C18:0.

The proportions of C17:0 cyclo varied between strains Az-1, Az-2, and Az-3. It also varied between A. restricta SUA2T determined this study and previously reported data from Bae et al

(2007). Previous studies showed an increase in C17:0 cyclo in Pseudomonas strains as cells entered late stationary (LS) or stationary growth in fatty acid methyl ester (FAME) profiles determined using the MIDI system. Variability of this cellular fatty acid has also been noted for other Pseudomonas spp. in stationary phase. Slight changes in growth conditions or cell growth stages can create variability between C17:0 cyclo (Haack et al., 1994).

A. oryzae strains 6a3T and PS had similar abundances for each fatty acid. Major fatty

T acids present in these strains include C16:0 (50.8% for 6a3 and 50.1% for PS), Summed Feature

T T 3 (41 % for 6a3 and 40.2% for PS), and C18:1 ω7c (4.0% for 6a3 and PS). Fatty acids present in lower amounts (<2% each) for both strains were C8:0 3OH, C10:0 3OH, C14:0, C14:1 ω5c, C15:1 ω6c,

C17:0, C18:0. Previously reported cellular fatty acid data from Bae et al. (2007) varied minimally from the results in this study.

T Major fatty acids in A. restricta SUA2 were C16:0 (40.3%), Summed Feature 3 (37%),

C10:0 3OH (6 %), C18:1 ω7c (5.5%), C12:0 (3.3%), and C15:0 (2.7%). Previously reported cellular fatty acid data from Bae et al. (2007) also varied minimally from the results in this study. Fatty acids C14:0, C14:1 ω5c, C15:1 ω6c, C17:0 cyclo, C17:0, C18:1 ω9c, and C18:0 were present in lower amounts (<2% each). Overall, major cellular fatty acids detected in all strains were Sum in

Feature 3, C16:0, and C18:1 ω7c. A summary table presenting cellular fatty acids that represent more than 0.5% in all strains are shown in Table 3.3. below.

Table 3.3. Presence of Cellular Fatty Acids in All Strains (those that represent <0.5% in all strains were omitted). All data was reported in this study except those marked with an “*” which was reported by Bae et al. (2007).

Peak Reference Reference Reference Name Az-1 Az-2 Az-3 6a3T 6a3T* PS PS* SUA2T SUA2T* Straight- chain C12:0 ------3.3 3.8 C14:0 1.9 1.1 1.4 0.8 0.6 0.9 0.8 1.2 1.2 C16:0 52.6 48.8 50.8 50.8 43.9 50.1 48.6 40.3 37.4 C17:0 0.5 1.2 - 0.2 0.3 0.4 0.4 0.5 0.6 C18:0 1.0 1.0 0.9 0.5 0.6 0.4 0.5 0.4 0.6 C15:0 1.2 3.4 0.7 0.7 0.4 1.7 0.6 2.7 2.4 Unsaturated C14:1 ω5c - 0.2 0.4 0.6 0.3 0.2 C15:1 ω6c - 0.6 - 0.1 0.4 0.4 Summed Feature 31 34.1 30.3 32.4 41.0 43.6 40.2 41.8 37.0 32 C18:1 ω9c - 0.7 - - - 0.1 C18:1 ω7c 6.2 5.2 6.1 4.0 8.3 4.0 5.4 5.5 6.2 Hydroxy C8:0 3OH 1.3 0.8 1.2 1.1 1.6 1.1 0.9 - - C10:0 3OH - 0.3 0.5 0.1 - 0.1 0.1 6.0 6.5 Cyclo C17:0 cyclo 1.1 6.3 5.6 - - - - 0.4 6.6

1 Summed Feature 3 is comprised of C16:1 ω7c and/or C15:0 iso 2OH

After 7 days of incubation at 25°C, results were inconclusive regarding toluene

consumption for all strains in VL7 supplemented with 0.1 g/L pure toluene or 2%, v/v toluene in

heptamethylnonane. Toluene mass balance recovery was poor in all samples including abiotic

negative controls. Later measurements were not conducted due to the mandatory laboratory shut

down in March 2020 on account of the COVID-19 outbreak from SARS-CoV-2 (Rothan and

Byrareddy, 2020). Further research is needed to determine if the isolates or reference strains are

able to consume toluene or other aromatic hydrocarbons.

3.6. Discussion

From phylogenetic, chemotaxonomic, and phenotypic features obtained in this study, strains Az-1, Az-2, and Az-3 appear to be essentially identical and generally comply with the emended description of the genus Azospira from Bae et al. (2007). Cells of strains Az-1, Az-2, and Az-3, like A. oryzae 6a3T and PS and A. restricta SUA2T, were Gram-negative staining, curved rods. All strains could grow aerobically and could use nitrate as an electron acceptor under the incubation conditions specified in this research. When 2% (w/v) NaCl was added to

VME medium, none of the strains produced turbidity. All strains showed growth at the temperature range between 15 and 40°C and the pH range of 7 to 9. All six strains were unable to assimilate N-acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-glucose, D-maltose, D- mannitol, D-mannose, phenylacetic acid, potassium gluconate, or trisodium citrate and were negative for indole production, fermentation of D-glucose, hydrolysis of esculin, and the presence of arginine dihydrolase, protease, and β-galactosidase. They were all able to utilize β- hydroxybutyric acid and succinic acid but not putrescine. PHB storage granules were produced in all six strains during incubation. Major cellular fatty acids detected in all six strains were C16:0,

Summed Feature 3 (C16:1 ω7c and/or C15:0 iso 2OH), and C18:1 ω7c.

Bae et al. (2007) described representatives of the genus Azospira as Gram-negative staining, curved rods that grow well aerobically. They are variable with respect to nitrate and perchlorate as electron acceptors, heterotrophic, oxidase-positive, and catalase-positive. The optimum growth temperature is about 37°C, and optimal pH range is neutral. They produce polyhydroxybutyrate (PHB) storage granules. The major cellular fatty acids detected are C16:1

ω7c, C16:0, C18:1 ω7c (Bae et al., 2007). Results from polyphasic characterization reported in this thesis chapter comply with a handful of the shared characteristics described in the emended 66

genus description from Bae et al. (2007). Experiments were not performed to assess for spore formation, motility, microaerophilic growth, perchlorate utilization, or nitrogen fixation, however. The completion of these tests is necessary to compare properties of the new isolates with the genus description.

A group having 94.3% identity to strains Az-1, Az-2, and Az-3 belonged in the genus

Propionivibrio (Table 3.1.). Apart from having only around 94% 16S rRNA gene sequence similarity to the new isolates (lower than the similarity to Azospira spp.), members of the genus

Propionivibrio are strict anaerobes (Tanaka et al., 1990). In contrast, strains Az-1, Az-2, and Az-

3 grew under aerobic as well as nitrate-reducing conditions.

It is suggested that the new isolates, Az-1, Az-2, and Az-3, represent a novel species because 16S rRNA gene comparison revealed that the isolates shared less than 97% similarity to the most closely related type strain, A. oryzae 6a3T and A. restricta SUA2T, and also and possess multiple phenotypic differences. Specifically, A. restricta SUA2T had green to yellow translucent colonies, and the remaining strains had pink to salmon colored colonies. Strains Az-1, Az-2, and

Az-3 were catalase negative, whereas, A. oryzae strains 6a3T and PS and A. restricta SUA2T were catalase positive. Strain Az-1, Az-2, and Az-3 were urease positive while A. oryzae strains

6a3T and PS were urease negative. The optimal temperature range for growth was determined to be 30-40°C for strains Az-1, Az-2, and Az-3 and both A. oryzae strains but was determined to be

22-40°C for A. restricta SUA2T. Growth at 1% (w/v) NaCl was observed for strains Az-1, Az-2, and Az-3 and A. oryzae strains but not A. restricta SUA2T. Strains Az-1, Az-2, and Az-3 also had substrate utilization profiles that differed from reference strains as shown in Table 3.6.

Results for strains 6a3T, PS, and SUA2T reported in this study agreed with those reported by Bae et al. (2007). For example, cell lengths, widths, and shape (curved rods) were all consistent with the previously reported results. Colony morphologies (which were only reported for VM ethanol medium by Bae et al (2007)) were also consistent with previously reported data.

Deviations were observed, however, in a portion of the substrate utilization profile for strain

SUA2T reported by Bae et al. (2007), specifically for L-aspartic acid, L-proline, propionic acid, putrescine, and succinic acid. It should be noted that testing methods varied between studies which may have resulted in varying substrate utilization profiles. Specifically in this study, cells were grown in liquid VL7 supplemented with 0.5 g/L substrate rather than being assessed on

Biolog GN and GP MicroPlates.

Overall, there were multiple phenotypic, chemotaxonomic, and phylogenetic differences between strains Az-1, Az-2, and Az-3 and the reference strains in this study, supporting the notion that strains Az-1, Az-2, and Az-3 represent a new species within the genus Azospira.

Results also demonstrate that strains Az-1, Az-2, and Az-3 are essentially identical; however, a full genome comparison would confirm this with higher certainty. Phenotypic differences between all strains are summarized in Table 3.4. below.

Table 3.4. Phenotypic Differences between the new isolates and previously described Azospira spp.

Characteristic Az-1 Az-2 Az-3 A. oryzae A. oryzae A. restricta 6a3T ( = PS ( = DSM SUA2T ( = DSM 21223) 13638) DSM 18626) Cell dimensions 2.4 ± 0.6 ND* 2.1 ± 0.9 2 ± 0.3 × 0.4 ND 1.6 ± 0.2 × (µm) × 0.5 × 0.6 0.5 Catalase - - - + + + Urease + + + - - + Optimal 30-40°C 30-40°C 30-40°C 30-40°C 30-40°C 22-40°C Temperature Range Growth at 1% NaCl + + + + + - Utilization of: D,L-lactic - - - + + + acid α-ketoglutaric + + + + + - acid L-proline - - - - - + Acetic acid + + + + + - Propionic acid - - - + + + L-aspartic acid - - - + + + L-glutamic acid - - - + + - L-malic acid + + + + + -

CHAPTER 4. CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH 4.1. Conclusions

Research described in this thesis involved experiments aimed at isolating and characterizing bacteria from a toluene-producing enrichment culture derived from contaminated groundwater at a Louisiana Superfund Site. Cultivation techniques were implemented to isolate bacteria from various generations of the toluene-producing enrichment culture. Following multiple transfers of well isolated colonies on low nutrient solid media plates, 278 bacterial isolates (and later an additional 56 isolates) were tentatively identified based on sequencing partial 16S rRNA gene sequences and comparisons with public databases.

Bacteria isolated during the course of research described in this thesis grouped most closely with genera from the phyla Actinobacteria, Firmicutes, and Proteobacteria. A majority of isolates (71.3% of the total), were most closely affiliated with the genus Propionicimonas.

These were divided into two subgroups based on tentative identification, with the subgroups comprising 50.9% (group 7) and 20.4% (group 8) of the total isolates. About a quarter of the total isolates (25.1%) showed highest 16S rRNA gene sequence identity with representatives from the genus Azospira but with identities of only about 97% to the closest characterized strain

(Azospira oryzae strain 6a3T). Remaining isolates, representing less than 4% of the total isolated bacteria, were most closely related with the genera Bacillus, Micrococcus, Anoxybacillus,

Cellulosimicrobium, and Bradyrhizobium. Of the 56 additional isolates isolated from later cultivation attempts following PCR with Acidobacteria-specific primer sets, 66% of the isolates identified >99% to the genus Agrobacterium. The remaining identified closely with the genera

Bacillus and Rhizobium.

Species from all of the bacterial genera most closely related to the isolates from this study, except those most closely related to the genus Micrococcus, have been reported in the literature to be inhabitants of soil and groundwater environments. Representatives from the genus Micrococcus have been isolated from and reported to be inhabitants of the human skin microbiome (Kloos et al., 1974). The majority of isolates had partial 16S rRNA gene sequences that were >98.6% similar to previously described taxa. An exception, however, was the group of isolates identified most closely to the genus Azospira.

Based on 16S rRNA gene sequencing, none of the isolates obtained during the course of this thesis research belonged within the phylum Acidobacteria. It can be postulated that the

Acidobacteria-affiliated group that is hypothesized to be the direct toluene producer in the SR enrichment culture may not grow well on solid media (e.g., low nutrient media variants) or may be entirely unculturable in pure culture on solid media employed in the present study. It is also possible, however, that further isolation efforts using the media reported here could lead to recovery of a more diverse group of isolates including those affiliated with the phylum

Acidobacteria.

Because isolates related most closely to the genus Azospira had 96.9% identity to

Azospira oryzae strains 6a3T and PS and 95.1% identity to Azospira restricta SUA2T, the only two species within the genus reported to date, three strains were chosen for comparative testing to determine whether they may represent a new species.

Results of polyphasic comparative testing revealed that strains Az-1, Az-2, and Az-3 had essentially identical phenotypic properties and could be differentiated from both validly published species in the genus Azospira. Cells of Az-1, Az-2, and Az-3 were Gram-negative

staining, curved rods that were approximately 2 µm in length and 0.5 µm in diameter. The strains were oxidase positive but catalase negative. Strains Az-1, Az-2, and Az-3 grew with 1% (w/v)

NaCl but not with 2% (w/v) NaCl. Optimal temperature range for growth is between 30 and

40°C, and optimal pH is between 5.5 and 6. Strains were positive for the presence of urease enzyme and were able to use acetic acid, β-hydroxybutyric acid, a-ketoglutaric acid, L-malic acid, and succinic acid. PHB storage granules were produced in all strains over a 3 day incubation period. Nitrate was completely reduced after incubation in anoxic VL7 medium.

Major cellular fatty acids present in these strains included C16:0 (48.8 to 52.6%), the fatty acids identified by the Sherlock MIDI System as Sum in Feature 3 (C16:1 ω7c and/or C15:0 iso 2OH)

(30.3 to 34.1%), and C18:1 ω 7c (5.2 to 6.2% ). Further research is needed to ascertain the potential for strains to utilize toluene or other aromatic hydrocarbons.

When compared with representatives from the previously reported species in the genus

(A.oryzae strains 6a3T and PS and A. restricta SUA2T), there were numerous phenotypic differences between strains Az-1, Az-2, and Az-3 and the reference strains. These phenotypic differences, combined with low similarity in nearly complete 16S rRNA gene sequences, support the classification of Az-1, Az-2, and Az-3 as representatives of a new species.

4.2. Recommendations for future research

4.2.1. Recommendations for Research Performed in Chapter 2

Numerous additional media formulations could be explored in an attempt to isolate toluene-producing organisms on solid media. Sait (2008) found that strain Ellin345 could growth white turbid cultures in liquid VL55 media at 25°C using some organic acids, aromatics, sugars, sugar polymers, and amino acids as carbon sources. In addition, Campanharo et al. (2016)

observed growth of Subdivision 1 Acidobacteria on PSYA 5 at pH 5 with sucrose as a carbon source. Sucrose supported turbid growth of Ellin345 in liquid VL55 media (Sait, 2008), so additional experimentation could include the addition of sucrose or the specified carbon sources presented by Sait (2008) to solidified PA2D, VL55, or PSYA 5 adjusted to pH 5 to increase the cultivability of the Acidobacteria. The pH range for growth of Ellin345 in liquid VL55 media with glucose was from pH 4 to 6.5 (Sait, 2008). This could also be adjusted for new solid media since media pH ranged only from pH 5.5 to 7.2 in experiments reported in this thesis.

The concentration of NaCl added to solid media employed in the experiments described in chapter 2 were based on Sait (2008); however, antibiotic concentrations selected for testing were determined somewhat arbitrarily and may have been too high to allow growth of bacteria of interest. In future research, antibiotic addition could cover a concentration range in an effort to select for Acidobacteria Subdivision 1 bacteria. More knowledge about the bacterial community composition in the toluene-producing enrichment cultures such as by preparing 16S rRNA gene libraries on later generations of the culture could help assess which approaches may be beneficial for the cultivation of toluene-producing organisms.

Future experimentation should include expanding the number of media variants and incubation conditions tested (especially replicating those that yielded PCR products with the

Tol2 primer set), increase the number of colonies transferred, and the frequency of colony transfers. Maximizing the number of culturable bacteria identified would increase the likelihood of identifying Acidobacteria (if they are culturable on solid media).

Also, about 23% of the total colonies selected for sequencing appeared to be mixed cultures based on 16S rRNA gene sequences. It is possible that a toluene-producing organism

could have grown in conjunction with another organism. Colonies that appeared to be mixed cultures were not re-transferred which may have affected the outcome of results.

Additionally, research to further evaluate and optimize the Acidobacteria-specific primer sets and employ them in plate wash PCR has the potential to increase the efficiency and reliability of the screening process. Adjustments to the thermal protocol, the primer sequences, or the DNA template quantity could potentially reduce some of the false positives observed.

4.2.2. Recommendations for Research Performed in Chapter 3

Experiments performed in this thesis were sufficient to conclude that strains Az-1, Az-2, and Az-3 likely represent a novel species within the genus Azospira. Additional tests, however, could be performed to further assess the potential of the strains to utilize additional carbon sources (e.g., toluene), utilize different electron acceptors (e.g., nitrate and perchlorate), and explore other metabolic capabilities.

It is recommended that additional tests be performed to more accurately associate a genus with strains Az-1, Az-2, and Az-3. Bae et al. (2007) described phenotypic characteristics such as the lack of spore formation, motility by means of a single polar flagellum, the ability of strains to use perchlorate and nitrate as electron acceptors, and nitrogen fixation to be classifiers of members in the genus Azospira. Comparative tests to determine whether strains Az-1, Az-2, and

Az-3 possess characteristics that may group them with other related genera could also be performed by following methods in Tanaka et al. (1990), Liu et al. (2017), and Imhoff et al.

(1984).

Additional experiments to reassess the ability of strains to reduce nitrate could be performed in liquid R2A medium prepared anoxically and supplemented with nitrate. Parallel

bottles could also be prepared with the addition of nitrite rather than nitrate. Strains grew in R2A within 2 to 3 days, so nitrate and nitrite measurements could be observed over the course of a much shorter time period than the 49 days employed in the present study (with long incubation due to lab closure during the global COVID-19 pandemic). Inoculated negative controls should also be included to account for growth on yeast extract or additional carbon sources.

Literature reported that A. oryzae strain PS was able to utilize perchlorate as an electron acceptor in addition to oxygen and nitrate. A. oryzae 6a3T and A. restricta SUA2T were unable to utilize perchlorate as an electron acceptor (Bae et al., 2007). Experiments to assess the ability of strains Az-1, Az-2, and Az-3 to use perchlorate could be assessed in a procedure similar to the nitrate and nitrite reduction tests described above, with inoculated anoxic liquid R2A medium supplemented with perchlorate observed for turbidity after incubation.

Nitrogen fixation was assessed by Bae et al. (2007) for A. oryzae strains 6a3T and PS and

T A. restricta SUA2 . All strains were able to fix nitrogen (N2 gas) indicated by the reduction of acetylene to ethylene. Research in this thesis did not explore the ability of new isolates to fix nitrogen. Experiments to observe nitrogen fixing ability could be performed in semi-solid, nitrogen-free SM medium (Reinhold et al., 1986). The gas headspace would be supplied with acetylene and incubated. Nitrogen fixation would be measured by observing acetylene reduction and ethylene production using gas chromatography. The presence of the nifH gene which could be determined using gene-specific primers in PCR or could be determined from a complete genome sequence could serve as an additional indicator for nitrogen fixation abilities.

A goal of this research was to assess the potential for isolates to contribute to toluene production, consumption, or transformation. Experiments described in chapter 2 were performed to determine if a subset of isolates were able to produce toluene in anaerobic PA2D medium.

After 3 weeks of incubation, no toluene was detected in any samples. A second experiment including the new Azospira-related isolates Az-1, Az-2, and Az-3 and the reference strains was performed following the same procedure, but no toluene was detected after 30 days. Additional experiments could be performed to measure potential toluene production after a longer incubation period (e.g., 60 days) as the anaerobic process may be slow. Positive controls and abiotic negative controls should be included to ensure acceptable media or indicate potential contamination.

Preliminary experiments to assess the ability of strains Az-1, Az-2, and Az-3 to utilize toluene were performed in this thesis research; however, mass balance recovery was very poor in both inoculated samples and abiotic negative controls, indicating that toluene may have been lost due to abiotic processes. This experiment could be repeated with steps implemented to mitigate abiotic losses of toluene (e.g., by minimizing penetration of septa). Multiple replicates prepared to allow for toluene measurements at multiple time steps without the need for repeated sampling from the same bottle would likely be beneficial. Also, implementing a range of initial toluene concentrations could help better assess the range of conditions for growth of strains with toluene

(if any). In experiments described in this thesis, a single concentration of 100 mg/L toluene was supplemented into anoxic VL7 media, and the relatively high concentration may have been toxic to the strains. Also, toluene supplied in the carrier heptamethylnonane may have been inhibitory or toxic to the strains as well. Further research could assess other potential carriers that may be more appropriate.

Genome sequencing is recommended for strains Az-1, Az-2, and Az-3. This would allow assessment of whether there is genetic variation between the strain, would enable genome-wide comparison for taxonomic classification purposes, and could facilitate identification of nitrogen

fixation genes (nifH) or genes associated with toluene production (PhdA and PhdB, as described by Beller et al., 2018). During the course of research described in chapter 3, high molecular weight DNA was prepared for the strains; however, it has not yet been employed for use in genome sequencing due to laboratory closures during the COVID-19 pandemic.

APPENDIX A. REPRESENTATIVE SEQUENCES FROM ISOLATES

The following assembled 16S rRNA gene sequences in fasta format are described in section 2.4.3 of this thesis.

1. Group 1: representative sequence MM 5.5-2c (1-2c) ACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGA ACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGG CGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTATGGGATTGGCTAAACCTCG CGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAG GGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACC TTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGG ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTC TGTCCCCCGAAGGGGAACGCCCTATCTCTAGGGTTGGCAGAGGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC CCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTA ATGCGTTTGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTT TACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTC AGCGTCAGTTACAGACCAAAGAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTA CGCATTTCACCGCTACACGTGGAATTCCGCTCTTCTCTTCTGCACTCAAGTTCCCCAG TTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGGAACCG CCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACC GCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCG GCAGTTACTCCGGTACTTGTTCTTCCCTAACAACAGAGTTTTACGATCCGAAAACCTT CATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTAC TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCT CTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAAT GCGCCGCGGGCCCATCTATAAGTGATAGCCGAAACCATCTTTCAGCTTTCTCTCATG TGAGAAAAAGCATTATCCGGTATTAGCTCCGGTTTCCCGAAGTTATCCCAGTCTTAT AGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCT 2. Group 2: Representative Sequence MM7-7c (2-7c) CAAGGGTTAGGCCACCGGCTTCGGGTGTTACCAACTTTCGTGACTTGACGGGCGGTG TGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGA CTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTG GGATTAGCTCCACCTCACAGTATCGCAACCCATTGTACCGGCCATTGTAGCATGCGT GAAGCCCAAGACATAAGGGGCATGATGATTTGACGTCGTCCTCACCTTCCTCCGAGT TGACCCCGGCAGTCTCCCATGAGTCCCCACCATTACGTGCTGGCAACATGGAACGAG GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAAC CATGCACCACCTGTGAACCCGCCCCAAAGGGGAAACCGTATCTCTACGGCGATCGA GAACATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTC

CGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCC CCAGGCGGGGCACTTAATGCGTTAGCTGCGGCGCGGAAACCGTGGAATGGTCCCCA CACCTAGTGCCCAACGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTC CCCATGCTTTCGCTCCTCAGCGTCAGTTACAGCCCAGAGACCTGCCTTCGCCATCGG TGTTCCTCCTGATATCTGCGCATTCCACCGCTACACCAGGAATTCCAGTCTCCCCTAC TGCACTCTAGTCTGCCCGTACCCACCGCAGATCCGGGGTTAAGCCCCGGACTTTCAC GACAGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAATAATTCCGGATAACGC TCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTGC AGGTACCGTCACTTTCGCTTCTTCCCTACTGAAAGAGGTTTACAACCCGAAGGCCGT CATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCAC TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCT CTCAGGCCGGCTACCCGTCGTCGCCTTGGTGAGCCATTACCTCACCAACAAGCTGAT AGGCCGCGAGTCCATCCAAAACCGATAAATCTTTCCAACACCCACCATGCGGTGGA CGCTCCTATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCAGAGTTAAGGGCAGG TTACTCACGTGTTACTCACCCGTTCGCCACTAATCCACCCAGCAA Sequence 7-21 was slightly different but classified in the same group- differences shown in table 3. Group 3: Representative Sequence VL55 AX-15c (9-15c) *27f Forward Sequence Only; Assembly was unavailable due to quality of reverse sequence (with 1492r primer) GCTTCTGTTTGGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCCGTAAG ACGGGGATAACTTCGGGAAACCGGAGCTAATACCCGATAACCCTGAAGACCGCATG GTCTTTAGTTGAAAGGCGGCTTCGGCTGTCACTTACGGATGGGCCCGCGGCGCATTA GCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGG GTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGT AGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGCGAAGA AGGTCTTCGGATTGTAAAGCTCTGTTGTTAGGGAAGAACAAGTATGGTTCGAATAGG GCCGTACCTTGACGGTACCTAACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCC GCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGC AGGCGGTTCCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGG AAACTGGGGGACTTGAGTGCAGAAGAGGAGAGCGGAATTCCACGTGTAGCGGTGAA ATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACT GACGCTGAGGCGCGAAAGCG 4. Group 4: Representative Sequence VL6.5-23cii (7-23cii) ATGGGCTTCGGGTGTTACCGACTTTCGTGACTTGACGGGCGGTGTGTACAAGGCCCG GGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCATG GGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTGGGATTCGCTCCAC CTTACGGTATCGCAGCCCTTTGTACCGGCCATTGTAGCATGCGTGAAGCCCAAGACA TAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGT CTCCCATGAGTCCCCGGCATAACCCGCTGGCAACATGGGACGAGGGTTGCGCTCGTT GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTG TGCACGAGTGTCCAAAGAGACCACCATCTCTGGTGGCTTCTCGTGCATGTCAAGCCT TGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGG 79

CCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCACTT AATGCGTTTGCTGCGGCACGGAACTCGTGGAATGAGCCCCACACCTAGTGCCCAAC GTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCATGCTTTCGCTCC TCAGCGTCAGTTGCGGCCCAGAGACCTGCCTTCGCCATCGGTGTTCCTCCTGATATC TGCGCATTCCACCGCTACACCAGGAATTCCAGTCTCCCCTACCGCACTCTAGTCTGC CCGTACCCGATGCAAGCTCGAGGTTGAGCCTCGAGTTTTCACACCAGACGCGACAA ACCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTAT TACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTG CGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCTTCATCCCTCACGCGGC GTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGG AGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCC GTCGTCGCCTTGGTAGGCCATCACCCCACCAACAAGCTGATAGGCCGCGAGCCCATC CCTGACCGAAAAACTTTCCAACCACCCCCATGCGAGGACGGCTCATATCCGGTATTA GCCCCGGTTTCCCGGAGTTATCCCGAAGTCAAGGGCAGGTTACTCACGTGTTACTCA CCCGTTCGCCACTAATCCGCCCAGCAAGCTGGGCATCATCGTTCGAC 5. Group 5: Representative sequence OVL55-24c (11-24c); Assembled sequence is short because the first ~200 bp showed overlap GGGTCGCCCCTTAGCATCCCATTGTCACCGCCATTGTAGCACGTGTGTAGCCCAGCC CGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCGCGGCTTATCACCGGC AGTCTCCTTAGAGTGCTCAACTAAATGGTAGCAACTAAGGACGGGGGTTGCGCTCGT TGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCT GTCTCCGGTCCAGCCGAACTGAAGAACTCCGTCTCTGGAGTCCGCGACCGGGATGTC AAGGGCTGGTAAGGTTCTGCGCGTTGCGTCGAATTAAACCACATGCTCCACCGCTTG TGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGG AATGCTTAAAGCGTTAGCTGCGCCACTAGTGAGTAAACCCACTAACGGCTGGGCATT CATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCG TGCCTCAGCGTCAGTATCGGGCCAGTGAGCCGCCTTCGCCACTGGTGTTCTTGCGAA TATCTACGAATTTCACCTCTACACTCGCAGTTCCACTCACCTCTCCCGAACTCAAGAT CTTCAGTATCAAAGGCAGTTCTGGAGTTGAGCTCCAGGATTTCACCCCTGACTTAAA GACCCGCCTACGCACCCTTTACGCCCAGTGATTCCGAGCAACGCTAGCCCCCTTCGT ATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTATTCTTGCGGTACCGTCATT ATCTTCCCGCACAAAAGAGCTTTACAACCCTAGGGCCTTCATCACTCACGCGGCATG GCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTA 6. Group 6: Representative Sequence Az-3 (SR6.5-41b); Trimmed assembly ATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGAGCTT GCTCCTGGTGGCGAGTGGCGAACGGGTGAGTAATGCATCGGAACGTATCCTGGAGT GGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATATTCTGTGAGCAGGAAAGC GGGGGACCTTCGGGCCTCGCGCTCTTGGAGCGGCCGATGTCGGATTAGCTAGTTGGT GAGGTAAAGGCTCACCAAGGCGACGATCCGTAGCAGGTCTGAGAGGATGATCTGCC ACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTT GGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCGG GTTGTAAAGCTCTTTCGCGAGGGAAGAAATTGCACCGGATAATACCTGGTGTAGATG ACGGTACCTTGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACG TAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTCG

TAAGACAGGCGTGAAATCCCCGGGCTTAACCTGGGAACTGCGCTTGTGACTGCGAG GCTAGAGTACGGCAGAGGGAGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAG ATGTGGAGGAATACCGATGGCGAAGGCAGCCTCCTGGGCCAGTACTGACGCTCATG CACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAAC GATGTCAACTAGGTGTTGGTGGGGTTAAACCCATTAGTACCGCAGCTAACGCGTGAA GTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGA CCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACC TACCCTTGACATGCCAGGAACTTTCCAGAGATGGATTGGTGCCCGAAAGGGAGCCT GGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTGTCATTAATTGCCATCATTTAGTTGGGCACTTTAATG AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCC TTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACAAAGGGTTGCCAAGCCGC GAGGTGGAGCTAATCTCAGAAAGCCGATCGTAGTCCGGATTGCAGTCTGCAACTCG ACTGCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGCATGTCACGGTGAATACGT TCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTCTACCAGAAGTA GTTAGCCTAACCGTAAGGAGGGCGATTACCACGGTAGGATTCATGACTGGGGTG 7. Group 7: Representative Sequence VL6.5-40c (7-40c) TTCGGGTGTTACCGACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACG TATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCATGGGGTCG AGTTGCAGACCCCAATCCGAACTGAGACTGGCTTTATGGGATTCGCTCCACCTTGCG GTATTGCAGCCCTTTGTACCAGCCATTGTAGCATGCGTGAAGCCCTGGACATAAGGG GCATGATGACTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTTCTAT GAGTTCCCGGCCGAACCGCTGGCAACATAGAACGAGGGTTGCGCTCGTTGCGGGAC TTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTATACCG ACCTTGCGGGGCACCTGTCTCCAGATGTTTCCGGTATATGTCAAACCCAGGTAAGGT TCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCA ATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCACTTAATGCGTTA GCTTCGGCACGGAGTCCGTGGAAGGACCCCACACCTAGTGCCCACCGTTTACGGCGT GGACTACCAGGGTATCTAAGCCTGTTTGCTCCCCACGCTTTCGCTTCTCAGCGTCAG GAAATGTCCAGAGAACCGCCTTCGCCACTGGTGTTCCTCCTGATATCTGCGCATTCC ACCGCTCCACCAGGAATTCCGTTCTCCCCTACATCCCTCAAGTCTGCCCGTATCGAA AGCAGGCTCAGGGTTAAGCCCTGAGTTTTCACTTCCGACGCAACAGACCGCCTACAA GCTCTTTACGCCCAATAATTCCGGACAACGCTCGCACCCTACGTATCACCGCGGCTG CTGGCACGTAGTTAGCCGGTGCTTCTTCTGCAGGTACCGTCAGAAATCCTTCGTCCCT GCTGAAAGCGGTTTACAACCCGAAGGCCTTCATCCCGCACGCGGCGTTGCTGCGTCA GGCTTTCGCCCATTGCGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCG TATCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTT GGTGAGCCGTTACCTCACCAACAAGCTGATAGGCCGCGAGCCCATCCTTGACCGTCG GAGCTTTCCACCCCGGCAGATGCCTGCCAGGGTCGTATCCGGTATTAGCAGCTGTTT CCAACTGTTATCCCAGTGTCAAGGGTAGGTTGCTCACGTGTTACTCACCCGTTCGCC ACTGATCAGAGGAGCAAGCTCCTCGTCAC 8. Group 8: Representative Sequence VL55 GX-12c (8-12c) CAAGGGTTAGGCACCGGCTTCGGGTGTTACCGACTTTCATGACTTGACGGGCGGTGT GTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGAC

TCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACTGGCTTTATGG GATTCGCTCCACCTTGCGGTATTGCAGCCCTTTGTACCAGCCATTGTAGCATGCGTG AAGCCCTGGACATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGAGTT GACCCCGGCAGTCTTCTATGAGTTCCCGGCCGAACCGCTGGCAACATAGAACGAGG GTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCC ATGCACCACCTGTATACCGACCTTGCGGGGCACCTGTCTCCAAGTGTTTCCGGTATA TGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCG CTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGG CGGGGCACTTAATGCGTTAGCTACGGCACGGAATCCGTGGAAGGATCCCACACCTA GTGCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAAGCCTGTTTGCTCCCCACG CTTTCGCTTCTCAGCGTCAGGAAATGTCCAGAGAACCGCCTTCGCCACTGGTGTTCC TCCTGATATCTGCGCATTCCACCGCTCCACCAGGAATTCCGTTCTCCCCTACATCCCT CAAGTCTGCCCGTATCGAAAGCAGGCTCAGGGTTAAGCCCTGAGTTTTCACTTCCGA CGCAACAGACCGCCTACAAGCTCTTTACGCCCAATAATTCCGGACAACGCTCGCACC CTACGTATCACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTGCAGGTACC GTCAGAAAACCTTCGTCCCTGCTGAAAGCGGTTTACAACCCGAAGGCCTTCATCCCG CACGCGGCGTTGCTGCGTCAGGCTTTCGCCCATTGCGCAATATTCCCCACTGCTGCC TCCCGTAGGAGTCTGGG

APPENDIX B. STRAIN AZ-3 ASSEMBELED SEQUENCE

>Assembled nearly complete 16S rRNA Gene Sequence from strain Az-3 (as described in section 3.5)

ATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGAGCTT GCTCCTGGTGGCGAGTGGCGAACGGGTGAGTAATGCATCGGAACGTATCCTGGAGT GGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATATTCTGTGAGCAGGAAAGC GGGGGACCTTCGGGCCTCGCGCTCTTGGAGCGGCCGATGTCGGATTAGCTAGTTGGT GAGGTAAAGGCTCACCAAGGCGACGATCCGTAGCAGGTCTGAGAGGATGATCTGCC ACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTT GGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCGG GTTGTAAAGCTCTTTCGCGAGGGAAGAAATTGCACCGGATAATACCTGGTGTAGATG ACGGTACCTTGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACG TAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTCG TAAGACAGGCGTGAAATCCCCGGGCTTAACCTGGGAACTGCGCTTGTGACTGCGAG GCTAGAGTACGGCAGAGGGAGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAG ATGTGGAGGAATACCGATGGCGAAGGCAGCCTCCTGGGCCAGTACTGACGCTCATG CACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAAC GATGTCAACTAGGTGTTGGTGGGGTTAAACCCATTAGTACCGCAGCTAACGCGTGAA GTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGA CCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACC TACCCTTGACATGCCAGGAACTTTCCAGAGATGGATTGGTGCCCGAAAGGGAGCCT GGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTGTCATTAATTGCCATCATTTAGTTGGGCACTTTAATG AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCC TTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACAAAGGGTTGCCAAGCCGC GAGGTGGAGCTAATCTCAGAAAGCCGATCGTAGTCCGGATTGCAGTCTGCAACTCG ACTGCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGCATGTCACGGTGAATACGT TCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTCTACCAGAAGTA GTTAGCCTAACCGTAAGGAGGGCGATTACCACGGTAGGATTCATGACTGGGGTG

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VITA

Madison Mikes was born in New Orleans, Louisiana to Celeste and Steven Mikes. She attended and completed high school at The Academy of the Sacred Heart in New Orleans,

Louisiana in 2014 where she was the varsity soccer team captain. The following fall, she started college at Louisiana State University in Baton Rouge and worked as a part-time campus employee in the Department of French Studies. Alongside her studies as an Environmental

Engineer, she worked as a lifeguard for University Recreation. In her final year as an undergraduate at Louisiana State University, she began working in Dr. Moe’s research laboratory where she began conducting experiments in support of soil and groundwater remediation activities. This laboratory experience influenced her to apply to the Civil Engineering graduate program at LSU. In the fall of 2018, Madison began pursuing her master’s degree in Civil

Engineering with a concentration in Environmental Engineering. She performed her thesis research under the guidance of Dr. William Moe. She hopes to receive her master’s degree in

December 2020 and will begin her career in Environmental Engineering in New Orleans, LA.